WO2016187703A1 - Bacterial and fungal metabolites possessing anti-microbial activity against xanthomonas species, compositions, methods, kits and uses relating to same - Google Patents

Bacterial and fungal metabolites possessing anti-microbial activity against xanthomonas species, compositions, methods, kits and uses relating to same Download PDF

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Publication number
WO2016187703A1
WO2016187703A1 PCT/CA2016/050571 CA2016050571W WO2016187703A1 WO 2016187703 A1 WO2016187703 A1 WO 2016187703A1 CA 2016050571 W CA2016050571 W CA 2016050571W WO 2016187703 A1 WO2016187703 A1 WO 2016187703A1
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composition
ppm
xanthomonas
plant
species
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PCT/CA2016/050571
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French (fr)
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Fadi Dagher
Éric DEZIEL
Snizhana OLISHEVSKA
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Institut National De La Recherche Scientifique
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Priority to CA2985657A priority Critical patent/CA2985657A1/en
Priority to US16/070,703 priority patent/US11089785B2/en
Publication of WO2016187703A1 publication Critical patent/WO2016187703A1/en
Priority to US17/373,703 priority patent/US20210337806A1/en

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
    • A01N63/25—Paenibacillus
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/30—Microbial fungi; Substances produced thereby or obtained therefrom
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/10—Animals; Substances produced thereby or obtained therefrom
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
    • A01N63/22—Bacillus

Definitions

  • the present invention relates to anti-microbial metabolites from various bacterial and fungal species. More particularly, the present invention relates to bacterial and fungal metabolites having anti-microbial activity against Xanthomonas species.
  • Xanthomonas is a genus of Gram-negative rod-shaped bacteria that acts as a plant pathogen. Many Xanthomonas species cause serious diseases in hundreds of plant hosts, including a wide variety of economically important crops, such as tomato, pepper, lettuce, strawberries, walnuts, rice, citrus, banana, cabbage, and bean.
  • copper-based formulations may also have a negative environmental impact, since copper ions are not degraded in soil and can accumulate to high levels at locations with a history of intensive copper application.
  • SAR Systemic Acquired Resistance
  • ASM acibenzolar-S-methyl
  • Biological control of plant diseases may offer a safer and more specific effective alternative to the use of synthetic chemicals, and may pose less environmental concerns. There is thus a need for biological products for controlling plant pest diseases, such as Xanthomonas species.
  • the present inventors have unexpectedly discovered anti-microbial metabolites secreted by various bacterial and/or fungal species that have anti-bacterial activity against Xanthomonas species, which cause disease in plant hosts, including a wide variety of crops. Bacterial and fungal species producing these metabolites were identified from environmental samples (seeds, different vegetable organs and soil) from different geographic locations. Compositions, methods, uses, and kits relating to the identified anti-microbial metabolites are also described herein.
  • the present description relates to a composition comprising metabolites from a bacterial and/or fungal species, wherein the metabolites have antimicrobial activity against Xanthomonas species.
  • the metabolites may be extracellular bacterial and/or extracellular fungal metabolites (e.g., secondary metabolites).
  • the metabolites may be from a: Bacillus species; Paenibacillus species; Burkholderia species; Mortierella species; Giberella species; Fusarium species; Aspergillus species; Penicillium species; or any combination thereof.
  • the metabolites may be from: Paenibacillus polymyxa; Paenibacillus peoriae; Bacillus amyloliquefaciens; Burkholderia cepacia; Mortierella alpine; Giberella moniliformis; Fusarium oxysporum; Aspergillus n/ger Tiegh; Aspergillus hiratsukae; Penicillium ochrochloron; or any combination thereof.
  • the metabolites may be from Burkholderia cepacia, Paenibacillus polymyxa, Paenibacillus peoriae, and/or Bacillus amyloliquefaciens.
  • the metabolites may be from Paenibacillus peoriae. In some embodiments, the metabolites may be from Bacillus amyloliquefaciens. In some embodiments, the metabolites may be from Paenibacillus polymyxa T1 B; Paenibacillus polymyxa 44; Paenibacillus sp. 62; Paenibacillus polymyxa 273 (since renamed as Paenibacillus peoriae 273); Paenibacillus polymyxa 329; Paenibacillus sp.
  • Paenibacillus polymyxa 390 Paenibacillus polymyxa To99 (since renamed as Paenibacillus peoriae To99; deposited on March 9, 2015 at the NRRL under No NRRL B-67020); Paenibacillus polymyxa TP12; Paenibacillus polymyxa TP29; Paenibacillus polymyxa TP77; Paenibacillus polymyxa V25T; Paenibacillus polymyxa TFr60; Paenibacillus sp.
  • TFr101 since renamed as Paenibacillus peoriae TFr101 ; deposited on March 9, 2015 at the NRRL under No NRRL B-67019; Paenibacillus polymyxa TAu1 ; Paenibacillus polymyxa TM54; Bacillus amyloliquefaciens subsp. plantarum 16; Bacillus amyloliquefaciens subsp. plantarum 33; Bacillus amyloliquefaciens subsp. plantarum 71 (deposited on March 9, 2015 at the NRRL under No NRRL B-67021); Bacillus amyloliquefaciens subsp.
  • the metabolites may be from: Bacillus amyloliquefaciens subsp. plantarum 335; Bacillus amyloliquefaciens subsp. plantarum VFb49; Burkholderia cepacia BC19; Burkholderia cepacia BC153; Mortierella sp. VFb1 ; Giberella sp. TFr4; Fusarium sp. FI3S; Aspergillus sp. 8PT; Aspergillus sp. FG; Penicillium sp. VFr37; or any combination thereof.
  • the metabolites may be from: Bacillus amyloliquefaciens subsp.
  • the metabolites may have antimicrobial activity against phytopathogenic Xanthomonas species.
  • the metabolites may have antimicrobial activity against Xanthomonas campestris, Xanthomonas perforans, Xanthomonas gardneri, or any combination thereof.
  • the Xanthomonas campestris may comprise Xanthomonas campestris MAPAQ #901 and/or Xanthomonas campestris ED1985, or the Xanthomonas gardneri may comprise Xanthomonas gardneri DC00T7A.
  • the Xanthomonas perforans may comprises Xanthomonas perforans T1 , T2, T3, T4, T5, or any combination thereof.
  • the Xanthomonas perforans may comprise Xanthomonas perforans T4.
  • the metabolites may further have antimicrobial activity against a plant and/or a human pathogenic microorganism.
  • the pathogenic microorganism may be a virus, bacteria, fungus (including a microscopic fungus), yeast, mold, or any combination thereof.
  • the pathogenic microorganism may be: Xanthomonas euvesicatoria; Xanthomonas fraganae; Xanthomonas perforans; Xanthomonas campestns; Xanthomonas gardneri; Pseudomonas syringae; Erwinia amylovora; Burkholderia glumae; Escherichia coli; Bacillus subtilis; Staphylococcus aureus; Pseudomonas aeruginosa; or any combination thereof.
  • the pathogenic microorganism may be: Xanthomonas euvesicatoria R4; Xanthomonas gardneri DC00T7A; Xanthomonas fraganae LMG 708; Pseudomonas syringae DC3000; Erwinia amylovora 435; Burkholderia glumae LMG10905; Escherichia coli 0157:H7 EDL933; Bacillus subtilis ED66; Staphylococcus aureus ED711; Pseudomonas aeruginosa PA416A; or any combination thereof.
  • the antimicrobial activity may comprise anti-bacterial and/or anti-fungal antagonistic activity.
  • the metabolites may exhibit higher antimicrobial activity against Xanthomonas species, as compared to other species.
  • the metabolites may comprise lipopeptides and/or siderophores having anW-Xanthomonas activity.
  • the lipopeptides and/or siderophores may be from Bacillus and/or Paenibacillus.
  • the lipopeptides may be non-ribosomal lipopeptides (NRPs).
  • the lipopeptides and/or siderophores may comprise surfactin, fengycin, plipastatin, iturin, bacilysin, bacillibactin, bacillomycin, locillomycin, paenilarvin, pelgipeptin, polymyxin, paenibacterin, fusaricidin, bacitracin, tridecaptin, or any combination thereof.
  • the metabolites may comprise plipastatin and/or locillomycin.
  • the composition defined herein may further comprise an agriculturally acceptable excipient.
  • the composition defined herein may further comprise one or more of: non-toxic carriers, surfactants, preservatives, nutrients, UV protectants, stickers, spreaders and chelating agents.
  • the composition may lack viable cells from the bacterial and/or fungal species.
  • the composition may be a cell-free composition.
  • the composition may be a cell- free supernatant.
  • the composition may comprise killed cells from the bacterial and/or fungal species.
  • the composition may lack viable cells from the bacterial and/or fungal species.
  • the composition may comprise viable cells from the bacterial and/or fungal species.
  • the composition may comprise viable cells from the bacterial and/or fungal species.
  • the composition may comprise spores from the bacterial and/or fungal species.
  • the composition may be in the form of a liquid, concentrate, powder, tablet, gel, pellets, granules, or any combination thereof.
  • the composition once applied to a target plant, may have no detectable phytotoxic effect on the target plant, or on the fruits, nuts, or leaves thereof.
  • the composition may comprise at least 100 ppm, 200 ppm, 300 ppm, 400 ppm, 500 ppm, 600 ppm, 700 ppm, 800 ppm, 900 ppm, 1000 ppm, 1500 ppm, 2000 ppm, 2500 ppm, 3000 ppm, 3500 ppm, 4000 ppm, 4500 ppm, 5000 ppm, 5500 ppm, 6000 ppm, 6500 ppm, 7000 ppm, 8000 ppm, 8500 ppm, 9000 ppm, or 9500 ppm of the metabolites.
  • the composition may comprise between about 100 ppm, 200 ppm, 300 ppm, 400 ppm, 500 ppm, 600 ppm, 700 ppm, 800 ppm, 900 ppm, 1000 ppm, 1500 ppm, 2000 ppm, 2500 ppm, 3000 ppm, 3500 ppm, 4000 ppm, 4500 ppm, 5000 ppm, to about 10 000 ppm of the metabolites.
  • the compositions defined herein may be for use as an anti-microbial agent against a plant and/or human pathogenic microorganism.
  • the composition as defined herein may be a biopesticide.
  • the present description relates to the use of the compositions as defined herein as an antimicrobial agent against a plant and/or human pathogenic microorganism. In some aspects, the present description relates to the use of the compositions as defined herein for the manufacture of an anti-microbial agent against a plant and/or human pathogenic microorganism.
  • the pathogenic microorganism may be a Xanthomonas species.
  • the compositions or uses defined herein may be for application to a growing plant. In some embodiments, the growing plant may be a fruit plant, nut, cereal, vegetable, or flower.
  • the fruit may be: apple, apricot, banana, blackberry, blueberry, cantaloupe, cherry, cranberry, currant, grapes, greengage, gooseberry, honeydew, lemon, mandarin, melon, orange, peach, pears, pineapple, plum, raspberry, strawberry, tomatoes, watermelon, grapefruit, pepper, olive, or lime.
  • the nut may be: almond, beech nut, Brazil nut, butternut, cashew, chestnut, chinquapin, filbert, hickory nut, macadamia nut, pecan, walnut, or pistachio.
  • the cereal may be: amaranth, breadnut, barley, buckwheat, canola, corn, fonio, kamut, millet, oats, quinoa, cattail, chia, flax, kahiwa, pitseed goosefoot, wattleseed, rice, rye, sorghum, spelt, teff, triticale, wheat, or colza.
  • the vegetable may be: artichoke, bean, beetroot, broad bean, broccoli, cabbage, carrot, cauliflower, celery, chicory, chives, cress, cucumber, kale, dill, eggplant, kohlrabi, lettuce, onion, pepper, parsnip, parsley, pea, potato, pumpkin, radish, shallot, soybean, spinach, turnip, or peanut.
  • the growing plant may be a tomato plant; a pepper plant; a berry plant; a strawberry plant; lettuce; a citrus plant; a walnut plant; a rice plant; and/or a kiwi plant.
  • the flower may be: a species of the Euphorbiaceae; Euphorbia pulcherrima (poinsettia), Euphorbia milii (crown-of- thorns), Codiaeum variegatum (croton); a member of the family Rosaceae (Rosoideae/Rosa), Begoniaceae (Begonia), Araceae; Dieffenbachia, Anthurium, Philodendreae (Philodendron), Caladieae (Syngonium), English ivy or another Araliaceae species; Pelargonium (geranium), Ficus, Hydrangea, Zinnia, ornamental Prunus species, ornamental Peppers, or another flower and/or ornamental plant susceptible to infection by Xanthamonas.
  • compositions or uses defined herein may be for application to a plant cell or tissue which may be: a leaf, a stem, a flower, a fruit, a tuber, a rhizome, a corm, a root, or any combination thereof.
  • the present description relates to a method for producing a composition as defined herein, the method comprising culturing viable cells from the bacterial and/or fungal species to produce the metabolites; and harvesting the metabolites produced therefrom.
  • the viable cells may be cultured in Landy medium or Tryptic Soy Broth (TSB).
  • TTB Tryptic Soy Broth
  • the present description relates to an antimicrobial composition produced by a method defined herein.
  • the present description relates to a method for controlling the growth of a pathogenic microorganism on a target plant or tissue; the method comprising contacting the target plant or tissue with a composition as defined herein.
  • the contacting comprising spraying, irrigating, painting, daubing, and/or fogging, onto and/or into the target plant or tissue, the target plant or tissue's hydroponic substrate, and/or the target plant or tissue's agricultural earth.
  • the present description relates to a kit for preparing an aqueous solution for use in controlling pathogens on a plant tissue of a growing plant, the kit comprising: (a) an organism as defined herein which produces metabolites having antimicrobial activity against Xanthomonas species, and/or a composition as defined herein; and (b) a suitable container.
  • the container may be a pouch, a tablet, or a bucket.
  • the kit may be for use in controlling infection by the pathogenic microorganism on a plant.
  • the pathogenic microorganism may be ⁇ Xanthomonas species.
  • isolates To99 and/or TFr101 may be still be referred to as Paenibacillus polymyxa, it is understood that they may also be referred to as Paenibacillus peoriae, while still referring to the same deposited organisms.
  • Figure 1 shows the results of a phylogenetic analysis of bacteria within the genus Xanthomonas and the related genera Xylella and Stenotrophomonas.
  • Figure 2 shows an example of how the isolated bacterial strains were screened to identify those having antagonistic activity against X. campestris ED740 (A) and X. campestris ED1985 (B). Halo boundaries are delineated by dotted circles.
  • Figure 3 shows antagonistic activity of the bacterial strain "To65” against X. campestris ED1985.
  • the halo boundary around the bacterial colony of strain "To65” is indicated with a dotted circle.
  • Figure 4 shows an example of how isolated fungal strains were screened to identify those having antagonistic activity against X. campestris ED1985.
  • the halo boundary around the fungal colony of strain "TAu20" is indicated with a dotted circle.
  • Figure 5 shows the antimicrobial activity of three bacterial strains against X. perforans T4: Bacillus amyloliquefaciens subsp. plantarum 71 ("71"); P. polymyxa TFr101 ("TFr101”); and P. polymyxa To99 ("To99”). Halo boundaries are delineated by dotted circles.
  • Figure 6 shows the antimicrobial activity of several fungal strains against X. perforans T4: (A) Penicillium sp. VFr37 ("VRf37"); and (B) Giberella sp. TFr4 ("TFr4"), Fusarium sp. FI3S (“FI3S”)). Halo boundaries are delineated by dotted circles.
  • Figure 7 shows the agarose gel electrophoresis profiles of a PCR-amplified 390-bp region of the recA gene in two bacterial isolates: "19" and "153".
  • "+” is Burkholderia cepacia ATCC 25416, "-” is a negative control.
  • Molecular sizes in base pairs of the DNA standard are indicated in the left-hand margin.
  • Figure 8 shows the bactericidal effect of a solution of coppenmancozeb (2:1) against Xanthomonas perforans T4.
  • the halo boundary is indicated with a dotted circle.
  • Figure 9 presents photographs showing the effects of different treatments on leaf spots on the leaves of tomato seedlings.
  • A not-infected and treated by water
  • B infected by X. perforans T4 and treated by Tryptic Soy Broth (TSB);
  • C & D infected by X. perforans T4 and treated by water - upper (C) and lower (D) leaves surfaces are shown;
  • E & F infected by X. perforans T4 and treated by copper plus mancozeb - upper (E) and lower (F) leaves surfaces are shown;
  • G & H infected by X. perforans T4 and treated by cell-free supernatant of B. amyloliquefaciens subsp.
  • plantarum 71 - upper (G) and lower (H) leaves surfaces are shown;
  • I & J infected by X. perforans T4 and treated by cell-free supernatant of P. polymyxa To99 - upper (I) and lower (J) leaves surfaces are shown;
  • K & L infected by X. perforans T4 and treated by cell-free supernatant of P. polymyxa TFr101 : upper (K) and lower (L) leaves surfaces are shown;
  • M not-infected and treated by cell-free supernatant of B. amyloliquefaciens subsp. plantarum 71 ; and
  • N not-infected and treated by cell-free supernatant of P.
  • Figure 10 shows the antimicrobial activity of Bacillus amyloliquefaciens subsp. plantarum 10-fold diluted cell-free supernatants against Xanthomonas species.
  • Figure 11 shows the antimicrobial activity of Paenibacillus polymyxa 10-fold diluted cell-free supernatants against Xanthomonas species.
  • Figure 12 shows a typical print of an untreated tomato leaf in panel (A), and one treated with B. amyloliquefaciens subsp. plantarum 71 in panel (B) after 24 hours of incubation.
  • Figure 13 shows microbial viability of bacterial isolates on tomato leaves as evaluated by the leaf print method. A whitish film on tomato leaves formed by Bacillus and Paenibacillus isolates 6 days after respective treatments (panels A, C, E, G, I), as well as leaf shape of their live bacterial colonies after leaf printing (panels B, D, F, H, J) are shown.
  • Figure 14 shows the quantity of CFUs per leaf at 1 h or 6 h following treatment of tomato leaves with live bacterial cells of the indicated Bacillus and Paenibacillus isolates, as compared to untreated tomato leaves.
  • Figure 15 shows the colonies formed on TSA by microorganisms isolated from untreated tomato leaves after 6 days (panel C), or from tomato leaves treated 6 days prior with B. amyloliquefaciens subsp. plantarum 71 (panel A) or P. polymyxa To99 (Panel B).
  • Figure 16 shows yellow-brownish lesions caused by X. perforans T4 on tomato leaves, susceptible reaction caused by X. gardneri DC00T7A manifested 10 days after infestation as well-defined brown spots appeared on leaves (panels A and B) and stems (panels C and D). Brown spot are indicated with arrows.
  • Figure 17 shows the preventative effect of pre-treating the leaves of tomato seedlings with live cells of Bacillus and Paenibacillus isolates, or their metabolites (supernatants), followed by infection with X. gardneri DC00T7A. Results are expressed as the quantity of spots per plant.
  • Figure 18 shows the results of agar disc diffusion assays performed in order to evaluate the sensitivity of bacterial metabolites (cell-free supernatants) to light exposure for 0 to 12 weeks. Antimicrobial activity was then tested weekly by placing the paper discs saturated with 10-fold diluted cell-free supernatants on a lawn of Xanthomonas gardneri DC00T7A and measuring the inhibition area (in mm 2 ).
  • A B. amyloliquefaciens subsp. plantarum 71 and VFb49;
  • B P. polymyxa To99, TFr101 , and 273.
  • Figure 19 shows the antimicrobial activities of Paenibacillus and Bacillus 10-fold diluted cell-free supernatants against X. gardneri DC00T7A after storage at -20°C for 0, 6 or 12 months.
  • Figures 20-23 show the results of four greenhouse trials that were conducted to determine the efficacy of metabolites and/or live bacterial strains to control bacterial leaf spots, as compared to standard chemicals agents.
  • the present invention relates to a composition
  • a composition comprising metabolites from a bacterial and/or fungal species, wherein said metabolites have antimicrobial (e.g., bactericidal) activity against Xanthomonas species.
  • antimicrobial e.g., bactericidal
  • the term "metabolites" refers to any compound, substance or by-product obtainable by the culture or fermentation of a microorganism as described herein.
  • the metabolites of the present description may be produced by culturing microorganisms and harvesting extracellular metabolites produced therefrom (e.g., released into the culture supernatants).
  • the metabolites of the present description may be produced using recombinant DNA technology (e.g., recombinant proteins).
  • the metabolite may be a proteinaceous substance (i.e., a substance comprising a linear polymer chain of at least 3 amino acids bonded together by peptide bonds), bacteriocins, lantibiotics, lipopeptides and/or polyketides.
  • the metabolites may be extracellular bacterial and/or extracellular fungal secondary metabolites.
  • secondary metabolites refers to compounds that are not directly involved in normal growth, development, or reproduction. Unlike primary metabolites, the absence of secondary metabolites does not result in immediate death, but rather in long-term impairment of survivability, fecundity or aesthetics.
  • the expression “antimicrobial” refers to the ability of the metabolites of the present description to prevent, inhibit, and/or destroy the growth of pathogenic microbes such as pathogenic bacteria and/or pathogenic fungi.
  • the expression “antimicrobial” encompasses agents or compounds exhibiting antagonistic activity against pathogenic microbes.
  • the antimicrobial activity may be in vitro antimicrobial activity or in vivo antimicrobial activity.
  • the present description relates to metabolites having antimicrobial activity against phytopathogenic Xanthomonas species.
  • pathogen or “pathogenic” refers to an organism capable of producing a disease in a plant or animal.
  • phytopathogen refers to a pathogenic organism that infects a plant.
  • Xanthomonas species refers to a microorganism belonging to the genus Xanthomonas (including pathogenic Xanthomonas species). Phylogenetic analysis of bacteria within the genus Xanthomonas and the related genera Xylella and Stenotrophomonas is shown in Figure 1. In some embodiments, "Xanthomonas species” includes the Xanthomonas species listed in Figure 1. Without being bound by theory, the virulence of Xanthomonas species may be due to their secretion of extracellular enzymes such as endoglucanases (e.g., carboxymethylcellulases) that hydrolyze cellulose. Xanthomonas species produce also high molecular weight exopolysaccharides (EPS) xanthan and effectors of type III secretion that contribute to its virulence (Ray et al., 2000).
  • EPS molecular weight exopolysaccharides
  • the metabolites of the present description may have antimicrobial activity against Xanthomonas campestris and/or Xanthomonas perforans.
  • the Xanthomonas campestris may comprise Xanthomonas campestris MAPAQ #901 and/or Xanthomonas campestris ED 1985.
  • Race T1 was identified as X. euvesicatoria
  • race T2 as X. vesicatoria
  • races T3, T4 and T5 were identified as Xanthomonas perforans (Jones et al., 2004).
  • Race T4 came about as a result of a mutation in the X. perforans avrXv3 gene, and has become prevalent and a major problem on tomato in the state of Florida (USA).
  • the metabolites of the present description may have antimicrobial activity against Xanthomonas perforans T1 , T2, T3, T4, T5, or any combination thereof.
  • the metabolites of the present description may have antimicrobial activity against Xanthomonas perforans T4.
  • new phylogenic analyses of bacterial and/or fungal species (and others) have resulted in taxonomic reclassifications. Such changes in taxonomic classification are within the scope of the present invention and, regardless of future reclassifications, a person of skill in the art would be able to identify the organisms of the present description using methods described herein and other methods within the capabilities of the skilled person.
  • the present description relates to metabolites which may be extracellular bacterial and/or extracellular fungal metabolites.
  • extracellular refers to the compounds that are secreted or released (either actively or passively) into the extracellular medium upon culture of viable cells, but may also include compounds that contact the extracellular medium, but which remain associated with the cell membrane.
  • the present description relates to metabolites from a: Bacillus species; Paenibacillus species; Burkholderia species; Mortierella species; Giberella species; Fusarium species; Aspergillus species; Penicillium species; or any combination thereof.
  • the expression "from a [genus] species” or “obtainable from a [genus] species” refers to a compound that may be obtained (i.e., that is obtainable) from the culture or fermentation of a species belonging to the recited genus, but does not necessarily mean that the metabolite must be obtained from that particular species or from the culture of a microorganism perse.
  • the present description relates to metabolites from an organism that is naturally- occuring and/or that has not been genetically modified using recombinant DNA technology, and thus qualifies as a natural biopesticide and/or natural bioproduct.
  • the present description relates to metabolites are from a: Bacillus species; Paenibacillus species; Burkholderia species; Mortierella species; Giberella species; Fusarium species; Aspergillus species; Penicillium species; or any combination thereof.
  • the present description relates to metabolites from: Paenibacillus polymyxa; Paenibacillus peoriae; Bacillus amyloliquefaciens; Burkholderia cepacia; Mortierella alpine; Giberella moniliformis; Fusarium oxysporum; Aspergillus niger Tiegh; Aspergillus hiratsukae; Penicillium ochrochloron; or any combination thereof.
  • the metabolites may be from Burkholderia cepacia, Paenibacillus polymyxa; Paenibacillus peoriae; and/or Bacillus amyloliquefaciens.
  • the metabolites may be from: Paenibacillus polymyxa T1 B; Paenibacillus polymyxa 44; Paenibacillus sp. 62; Paenibacillus polymyxa 273 (since renamed as Paenibacillus peoriae 273); Paenibacillus polymyxa 329; Paenibacillus sp.
  • Paenibacillus polymyxa 390 Paenibacillus polymyxa To99 (NRRL B-67020; since renamed as Paenibacillus peoriae To99); Paenibacillus polymyxa TP12; Paenibacillus polymyxa TP29; Paenibacillus polymyxa TP77; Paenibacillus polymyxa V25T; Paenibacillus polymyxa TFr60; Paenibacillus sp.
  • TFr101 (NRRL B- 67019; since renamed as Paenibacillus peoriae TFr101); Paenibacillus polymyxa TAu1 ; Paenibacillus polymyxa TM54; Bacillus amyloliquefaciens subsp. plantarum 16; Bacillus amyloliquefaciens subsp. plantarum 33; Bacillus amyloliquefaciens subsp. plantarum 71 (NRRL B-67021); Bacillus amyloliquefaciens subsp. plantarum 237; Bacillus amyloliquefaciens subsp.
  • the present description relates to metabolites that may have further antimicrobial, bactericidal, and/or fungicidal activity against a plant and/or a human pathogenic microorganism, such as a virus, bacteria, fungus, yeast, mold, or any combination thereof.
  • the antimicrobial activity may comprise antagonistic activity.
  • bactericidal or fungicidal refers to the ability of a composition or substance to increase mortality or inhibit the growth rate of bacteria or fungi, respectively.
  • the pathogenic microorganism may be: Xanthomonas euvesicatoria; Xanthomonas fraganae; Xanthomonas perforans; Xanthomonas campestns; Xanthomonas gardnen, Pseudomonas syringae; Erwinia amylovora; Burkholderia glumae; Escherichia coli; Bacillus subtilis; Staphylococcus aureus; Pseudomonas aeruginosa; or any combination thereof.
  • the pathogenic microorganism may be: Xanthomonas euvesicatoria R4; Xanthomonas gardneri DC00T7A; Xanthomonas fraganae LMG 708; Pseudomonas syringae DC3000; Erwinia amylovora 435; Burkholderia glumae LMG10905; Escherichia coli 0157:H7 EDL933; Bacillus subtilis ED66; Staphylococcus aureus ED711; Pseudomonas aeruginosa PA416A; or any combination thereof.
  • "mold” refers to a fungus that grows in the form of multicellular filaments called hyphae.
  • the present description relates to metabolites that may exhibit higher antimicrobial activity against Xanthomonas species, as compared to other species (e.g., non-Xanthomonas species).
  • the metabolites of the present description are specifically active against Xanthomonas species.
  • “specifically active” means that metabolites of the present description show relatively higher antimicrobial activity against Xanthomonas species, than phytopathogenic non-Xanthomonas species.
  • the present description relates to metabolites that may comprise lipopeptides and/or siderophores having anW-Xanthomonas activity such as lipopeptides and/or siderophores from Bacillus and/or Paenibacillus.
  • the lipopeptides may be non-ribosomal lipopeptides (NRPs).
  • the lipopeptides and/or siderophores may comprise one or more of surfactin, fengycin, plipastatin, iturin, bacilysin, bacillibactin, bacillomycin, locillomycin, paenilarvin, pelgipeptin, polymyxin, paenibacterin, fusaricidin, bacitracin, and tridecaptin.
  • the metabolites may comprise plipastatin and/or locillomycin.
  • the compositions of the present description may further comprise an agriculturally acceptable excipient.
  • the phrase "agriculturally acceptable excipien” refers to an essentially inert substance that can be used as a diluent and/or carrier for an active agent (e.g., antimicrobial metabolites of the present description) in a composition for treatment of plants.
  • the compositions of the present description may further comprise one or more of: non-toxic carriers, surfactants, preservatives, nutrients, UV protectants, stickers, spreaders and chelating agents.
  • the compositions of the present description may lack viable cells of the bacterial and/or fungal species from which the antimicrobial metabolites originate.
  • the compositions of the present description may be a cell-free composition (e.g., a cell-free supernatant). Cells can be removed by, for example, filtration and/or centrifugation.
  • the compositions of the present description may comprise killed cells from the bacterial and/or fungal species.
  • the compositions of the present description may comprise viable cells and/or spores from the bacterial and/or fungal species.
  • compositions of the present description may be in the form of a liquid, concentrate, powder, tablet, gel, paste, pellets, granules, or any combination thereof.
  • compositions of the present description once applied to a target plant, may have no detectable phytotoxic effect on the target plant, or on the fruits, nuts, or leaves thereof, as compared to a control plant that is untreated (e.g., treated with water or with another agent).
  • compositions comprise an effective amount of antimicrobial metabolites of the present description.
  • An "effective amount', as used herein, is an amount sufficient to effect beneficial or desired results.
  • An effective amount can be administered in one or more administrations. In terms of treatment, inhibition or protection, an effective amount is that amount sufficient to ameliorate, stabilize, reverse, slow or delay progression of the target infection or disease states.
  • the compositions may comprise at least 100 ppm, 200 ppm, 300 ppm, 400 ppm, 500 ppm, 600 ppm, 700 ppm, 800 ppm, 900 ppm, 1000 ppm, 1500 ppm, 2000 ppm, 2500 ppm, 3000 ppm, 3500 ppm, 4000 ppm, 4500 ppm, 5000 ppm, 5500 ppm, 6000 ppm, 6500 ppm, 7000 ppm, 8000 ppm, 8500 ppm, 9000 ppm, or 9500 ppm of the metabolites of the present description.
  • the compositions may comprise between about 100 ppm, 200 ppm, 300 ppm, 400 ppm, 500 ppm, 600 ppm, 700 ppm, 800 ppm, 900 ppm, 1000 ppm, 1500 ppm, 2000 ppm, 2500 ppm, 3000 ppm, 3500 ppm, 4000 ppm, 4500 ppm, 5000 ppm, to about 10 000 ppm of said metabolites of the present description.
  • the term "about' is used herein to indicate that a value includes the standard deviation of error for the device or method being employed to determine the value. In general, the terminology “about” is meant to designate a possible variation of up to 10%. Therefore, a variation of 1 , 2, 3, 4, 5, 6, 7, 8, 9 and 10% of a value is included in the term "about”.
  • compositions of the present description may be used as an anti-microbial (e.g., bactericidal and/or fungicidal) agent against a plant and/or human pathogenic microorganism, or for the manufacture of an anti-microbial agent for same.
  • the pathogenic microorganism may be a Xanthomonas species.
  • antimicrobial compositions, bacterial or fungal strains of the present description may be used for biological control.
  • biological control refers to the control of a pathogen or any other undesirable organism by the use of at least a second organism other than man.
  • the "host plant' in the context of biological control is the plant that is susceptible to disease caused by the pathogen.
  • the host plant In the context of isolation of an organism, such as a bacterium or fungal species, from its natural environment, the host plant is a plant that supports the growth of the bacterium or fungus, for example, a plant of a species the bacterium or fungus is an endophyte of.
  • the compositions defined herein may be a biopesticide or biological pesticide.
  • biopesticide and “biological pesticide” refer to non-naturally occurring commercial products that include naturally occurring metabolites/microorganisms which are formulated to have anti-microbial activity when applied to plants. Such formulations may increase the stability and/or concentrations of the metabolites/miroorganisms, which enable them to be useful as plant pesticides.
  • the present description relates to an isolated strain of antimicrobial bacterial or fungal strains as defined herein.
  • an "isolated" strain of a microbe is a strain that has been removed from its natural milieu. As such, the term “isolated” does not necessarily reflect the extent to which the microbe has been purified. But in different embodiments an "isolated" culture has been purified at least 2x, 5x, 10x, 50x or 100x from the raw material from which it is isolated.
  • a culture is isolated from soil as raw material, the organism can be isolated to an extent that its concentration in a given quantity of purified or partially purified material (e.g., soil) is at least 2x, 5x, 10x, 50x or 100x that in the original raw material.
  • a "substantially pure culture” of the strain of microbe refers to a culture which contains substantially no other microbes than the desired strain or strains of microbe. In other words, a substantially pure culture of a strain of microbe is substantially free of other contaminants, which can include microbial contaminants as well as undesirable chemical contaminants.
  • the expression "enriched culture” of an isolated microbial strain refers to a microbial culture wherein the total microbial population of the culture contains more than 50%, 60%, 70%, 80%, 90%, or 95% of the isolated strain.
  • the present description relates to genetically modified or mutant antimicrobial bacterial or fungal strains as defined herein.
  • mutant making reference to a microorganism refers to a modification of the parental strain in which the desired biological activity (e.g., ability to produce antimicrobial metabolites as defined herein) is similar to or higher than that expressed by the parental strain.
  • compositions of the present description may be applied to a growing plant, such as a fruit plant, nut, cereal, vegetable, or flower.
  • fruit plants include: apple, apricot, banana, blackberry, blueberry, cantaloupe, cherry, cranberry, currant, grapes, greengage, gooseberry, honeydew, lemon, mandarin, melon, orange, peach, pears, pineapple, plum, raspberry, strawberry, tomatoes, watermelon, grapefruit, pepper, olive, or lime.
  • nuts include: almond, beech nut, Brazil nut, butternut, cashew, chestnut, chinquapin, filbert, hickory nut, macadamia nut, pecan, walnut, or pistachio.
  • cereals include: amaranth, breadnut, barley, buckwheat, canola, corn, fonio, kamut, millet, oats, quinoa, cattail, chia, flax, kahiwa, pitseed goosefoot, wattleseed, rice, rye, sorghum, spelt, teff, triticale, wheat, or colza.
  • vegetables include: artichoke, bean, beetroot, broad bean, broccoli, cabbage, carrot, cauliflower, celery, chicory, chives, cress, cucumber, kale, dill, eggplant, kohlrabi, lettuce, onion, pepper, parsnip, parsley, pea, potato, pumpkin, radish, shallot, soybean, spinach, turnip, or peanut.
  • flowers include: species of the Euphorbiaceae (e.g., Euphorbia pulcherrima (poinsettia), Euphorbia milii (crown-of-thorns), Codiaeum variegatum (croton)), members of the family Rosaceae (RosoideaelRosa), Begoniaceae (Begonia), Araceae (e.g., Dieffenbachia, Anthurium, Philodendreae (Philodendron), and Caladieae (Syngonium), English ivy and other Araliaceae species, Pelargonium (geranium), Ficus, Hydrangea, Zinnia, ornamental Prunus species, ornamental Peppers, as well as other flowers and/or ornamental plants susceptible to infection by Xanthamonas.
  • Euphorbiaceae e.g., Euphorbia pulcherrima (poinsettia), Euphorbia milii (crown-of-thorns
  • compositions of the present description may be applied to a tomato plant, a pepper plant, a berry plant (e.g., strawberry plant), lettuce, a citrus plant, a walnut plant, a rice plant, a kiwi plant, or any combination thereof.
  • a tomato plant e.g., a pepper plant
  • a berry plant e.g., strawberry plant
  • lettuce e.g., a citrus plant
  • a walnut plant e.g., a walnut plant
  • rice plant e.g., a kiwi plant
  • compositions of the present description may be applied to a plant cell or tissue or organ obtained from: a leaf, a stem, a flower, a fruit, a tuber, a rhizome, a corm, a root, or any combination thereof, or a part thereof.
  • the present description relates to a method for producing a composition as defined herein, the method comprising culturing viable cells from a suitable bacterial and/or fungal species to produce the metabolites; and harvesting the metabolites of the present description produced therefrom.
  • culturing refers to the propagation of organisms on or in media of various kinds.
  • the culture media and/or culture/fermentation conditions may be modified and/or optimized to increase the anti-Xanthomonas activity of the metabolites that are produced.
  • Landy medium or variations thereof may be used to enhance the anti-Xanthomonas activity of metabolites produced by B. amyloliquefaciens (e.g., subsp. plantarum isolates 71 and VFb49).
  • Such medium may typically contain: glucose (e.g., 20 g/L), L- glutamic acid (e.g., 5.0 g/L), yeast extract (e.g., 1.0 g/L), K 2 HP0 4 (e.g., 1.0 g/L), MgS0 4 7H 2 0 (e.g., 0.5 g/L), KCI (e.g., 0.5 g/L), CuS0 4 (e.g., 1.6 mg/L), Fe 2 (S0 4 ) 3 (e.g., 1.2 mg/L), MnS0 4 (e.g., 0.4 mg/L).
  • glucose e.g., 20 g/L
  • L- glutamic acid e.g., 5.0 g/L
  • yeast extract e.g., 1.0 g/L
  • K 2 HP0 4 e.g., 1.0 g/L
  • MgS0 4 7H 2 0 e.g., 0.5 g
  • Landy medium or variations thereof may facilitate the production of polyketides and lipopeptides (Chen et al., 2009), as well as to enhance the production of biosurfactant (e.g., Ben Ayed, Jemil et al. 2015). Furthermore, without being bound by theory, Landy medium or variations thereof may increase the production of fusaricidin-family antibiotics (e.g., Vater et al., 2015) and/or siderophore bacillibactin (e.g., Li et al., 2014). In some embodiments, tryptic soy broth (TSB) (Kim et al., 2015) or variations thereof may be used to enhance the activity of metabolites produced by P.
  • TTB tryptic soy broth
  • polymyxa isolates e.g., To99, TFr101 and 273.
  • fermentation may be conducted at about 30°C (e.g., with shaking at 250 rpm).
  • optimization of the culture media and/or culture/fermentation conditions may improve the production and/or relative concentrations of lipopeptides (e.g., surfactin, fengycin, and/or iturin) and/or other metabolites having anW-Xanthomonas activity.
  • the present description relates to an antimicrobial composition produced by the aforementioned method.
  • the present description relates to a method for controlling the growth of a pathogenic microorganism on a target plant or tissue, the method comprising contacting the target plant or tissue with the composition of the present description.
  • the contacting may comprise spraying, irrigating, painting, daubing, and/or fogging, onto and/or into the target plant or tissue, the target plant or tissue's hydroponic substrate, and/or the target plant or tissue's agricultural earth.
  • the present description relates to a kit for preparing an aqueous solution for use in controlling pathogens on a plant tissue and/or plant cells of a growing plant, the kit comprising the composition as defined herein, and a suitable container.
  • the container may be a pouch, a tablet, or a bucket.
  • the kit may be used for controlling infection by the pathogenic microorganism (e.g., Xanthomonas species) on a plant.
  • compositions of the present description may be used or formulated with other antimicrobial agents, such as one or more of the agents described in Table 1.
  • NacillusProTM P. syringae pv. tomato
  • parabrevis strain N4 pv. vesicatoria, peppers, Bio InsumosNativa Bacillus subtilis strain Antibiosis, X. campestris pv. cucurbits, Ltda, chili N5, Bacillus cereus competition coralina, walnut, peanut, Valdes et al., 2012 strain N6, Bacillus Xanthomonas juglandis, hop, leafy
  • NogallTM Fruit, nut, and
  • Pathogenic strains used as indicators for evaluating antagonistic and antimicrobial activity of bacterial and fungal isolates are the following: Xanthomonas campestris ED1985 was isolated from Sherrington lettuce leaves. Xanthomonas campestris 901 was received from MAPAQ-Agri-Reseau-Phytoprotection. Xanthomonas perforans T4 and Xanthomonas euvesicatoria R4 were provided by University of Florida, Plant Pathology Faculty (USA). Xanthomonas gardneri DC00T7A was provided by Agriculture and Agri-Food Canada (London, ON, Canada).
  • Xanthomonas fragariae LMG 708 and Burkholderia glumae LMG10905 were received from the Belgian coordinated collections of microorganisms (BCCM). Erwinia amylovora 435 was provided by Institut detician et de developpement en agro architecture (IRDA, QC, Canada). Multi-drug resistant, clinical strains Pseudomonas aeruginosa PA416A and Staphylococcus aureus ED711, as well as Pseudomonas syringae DC3000 and Bacillus subtilis ED66 were used from the Deziel lab collection, INRS-lnstitut Armand-Frappier (Laval, QC, Canada). Escherichia coli 0157:H7 EDL933 was also tested. All strains are stored in 15% glycerol at -80°C. 1.4 First step of screening: Antagonistic activity assays
  • Antagonistic activity of bacterial isolates against Xanthomonas was determined by various Petri dish plate assays.
  • TSB at 30°C and resuspended in sterile water was spread on TSA plates to obtain a lawn of pathogenic bacteria.
  • bacterial strains were cultivated in 3 ml_ of TSB at 30°C,
  • the antimicrobial activity against X. perforans T4 was assessed using well-diffusion inhibition assay [Obradovic et al., 2002; WO/2012/051699; Lindow et al., 2003].
  • a lawn of indicator bacteria was produced on the surface of the agar plates.
  • Fifty ⁇ _ of X. perforans T4 (OD620 0.2) grown overnight in TSB at 30°C and resuspended in sterile water was spread on TSA plates.
  • Wells were bored into the agar layer with a sterile glass tube (10 mm diameter) and filled with 200 ⁇ _ of cell-free fermentation filtrate, taking care to avoid spillage on the surface of the medium. The plates were then incubated at room temperature (approx.
  • 612 bacterial and 124 fungal strains showed inhibition zones with different areas for different strains and were considered as possessing antagonistic activity against X. campestris ED740 and X. campestris ED1985 (e.g., Figure 2A, 2B, Figure 3, and Figure 4). All these isolates were stored in glycerol at -80°C. These strains were chosen for estimation of their cell-free filtrate activity against both phytopathogenic indicators.
  • Nonselective media R2A, Tryptic Soy Agar (TSA), Plate Count Agar (PCA).
  • Selective mec ia Burkholderia cepacia selective agar (BCSA), Benedict (for Steptomyces species), ⁇ Standard Error of Mean (SEM), * Diameter of well is 10 mm.
  • Paenibacillus polymyxa was identified as Paenibacillus polymyxa and one isolate was identified as Paenibacillus jamilae. All six isolates of the genus Bacillus were identified as B. amyloliquefaciens subsp. plantarum. Thus, not only Paenibacillus species but also Bacillus species were found to be active against pathogenic Xanthomonas bacteria.
  • fungi and other bacteria such as Burkholderia (Tables 3 and 4) were also found to be active against Xanthomonas species.
  • activity against Xanthomonas pathogenic strains is not limited to Bacillus and Paenibacillus species but it is most prevalent in these two species.
  • a 1.5 mL of overnight culture in TSB was pelleted in a microcentrifuge tube at 13000 rpm for 10 minutes. The supernatant was then discarded and the pellet was resuspended in 1 mL of extraction buffer (50 mM Tris-HCI, 5 mM EDTA, 3% SDS, pH 8). The resuspended cells were transferred in a microtube containing 150-200 mg of sterile glass beads. The tubes were then placed in the FastprepTM for 50 seconds at a 4 m/s speed, for two rounds and put on ice for 2 minutes between the rounds.
  • extraction buffer 50 mM Tris-HCI, 5 mM EDTA, 3% SDS, pH 8.
  • the broken cells were then centrifuged at 13000 rpm for 10 minutes and the supernatant was mixed with 1 :5 volume of ammonium acetate 10 N. The mixture was vortexed, placed on ice for 5 minutes, and centrifuged at 13000 rpm for 15 minutes at 4°C. The supernatant was placed on ice for 5 minutes and centrifuged again at 13000 rpm for 15 minutes at 4°C. The supernatant was then mixed with 1 : 1 volume of ice-cold isopropanol, and the DNA was precipitated for 1-2 hours at 4°C. The tubes were centrifuged at 13000 rpm for 15 minutes at 4°C and the supernatant was discarded.
  • PCR-amplifications were carried out in a 50- ⁇ _ reaction mixture (Table 6) containing Feldan Taq DNA Polymerase (Bio Basic Canada Inc., Markham, Ontario, Canada).
  • the amplifications were performed in a C1000 TouchTM Thermal Cycler (Bio-Rad Laboratory Inc., Canada) using specific PCR temperature protocol (Table 7).
  • PCR products were analyzed by agarose gel electrophoresis (1.0% of agarose, 100V, 60 min), DNA was stained by ethidium bromide (0.5 Mg/mL), and visualized under UV illumination.
  • This search tool was developed by the National Center for Biotechnology Information (NCBI) (US). If the sequence identity is above 99%, we could conclude that bacterial isolate belongs to the same species, and if the identity is higher than 97, strains are classified into the same genus or the same family.
  • a 1.6 kb 16S rRNA and 750 bp 18S rRNA and ITS regions fragments were amplified and sequenced from bacterial and fungal isolates respectively. Twenty-four bacterial and six fungal isolates were identified by gene sequencing (Table 8). Thus, using the BLAST alignment system, 2 bacterial isolates belong to the Burkholderia cepacia complex, 16 isolates belong to the Paenibacillus genus and 6 isolates belong to the Bacillus subtilis group.
  • the 6 fungal isolates were identified as Mortierella alpina Peyronel; Gibberella moniliformis Wineland; Fusarium oxysporum Schltdl; Aspergillus niger Tiegh; Neosartorya hiratsukae Udagawa, Tsub. & Y. Horie (Aspergillus hiratsukae); and Penicillium ochrochloron Biourge (Table 8).
  • Table 8 16S and 18S rRNA gene sequence identification of bacterial and fungal isolates
  • the rpoB gene was amplified using the primer pair rpoB1698f / rpoB2041 r (Table 12). PCR-amplifications were carried out in a 25- ⁇ _ reaction mixture (Table 13). The amplifications were performed using specific PCR temperature protocol (Table 14).
  • rpoB fragments were analyzed by agarose gel electrophoresis, stained by ethidium bromide and visualized under UV illumination.
  • BiologTM GENIII MicroPlate (OmniLog) was used. It allows analyzing a microorganism in 94 phenotypic tests: 71 carbon source utilization assays and 23 chemical sensitivity assays.
  • Several isolates from each group mentioned above were chosen for performing this test.
  • Well known Paenibacillus polymyxa and P. peonae strains (P. polymyxa ATCC7070, P. polymyxa CR1 , P. peoriae LMG 16104, P. peonae LMG 16111), as well as type strains: P. polymyxa LMG 13294, P. peoriae LMG 14832 and P. jamilae LMG 21667 were used for comparison with our isolates.
  • the isolates to be identified were grown on TSA and then resuspended in a special gelling inoculating fluid. Then, bacterial cell suspension was inoculated into the GENIII MicroPlate and incubated at 34 °C for 24 h to allow the phenotypic fingerprint to form. After incubation, the phenotypic fingerprints of purple wells were compared within isolates, known Paenibacillus polymyxa and P. peoriae strains and type strains of these species.
  • Bacterial isolates 329 and 273 showed the same similarity (96.8%) to P. polymyxa CR1 and P. jamilae LMG 21667 (Table 17). Due to these differences, fatty acid analyses were performed for further bacterial isolate identification.
  • Paenibacillus polymyxa 94.7%
  • the percentage of similarity is calculated as follows: (number of similar characteristics/total number of tests (94)) ⁇ 00.
  • Fatty acids analysis was performed to confirm the identification of the Paenibacillus isolates (Keystone Labs Inc., Edmonton, Alberta, Canada).
  • the fatty acids analysis is accomplished with the MIDITM method (which analyzes fatty acid methyl esters in bacterial samples by gas chromatography) and then compared with the Sherlock libraries consisting in more than 100,000 analyses of strains obtained from experts and from culture collections (www.midi- inc.com).
  • Microbial identification results from the MIDI system are expressed as a similarity index.
  • the similarity index (SI) is a numerical value which is an expression of the relative distance from the population mean. An exact match of the fatty acid makeup of the unknown and the mean of the library entry would result in a SI of 1.000. As each fatty acid varies from the mean percentage, the SI will decrease in proportion to the cumulative variance between the composition of the unknown and the library entry.
  • the rpoB, gyrA and gyrB gene fragments were used as molecular diagnostic markers to specifically identify bacterial isolates within the Bacillus subtilis group.
  • specific primers for amplification of each gene were used (Table 19).
  • PCR-amplifications were carried out in a 25- ⁇ _ reaction mixture (Table 20).
  • the amplifications were performed using specific PCR temperature protocol (Table 21).
  • rpoB, gyrA and gyrB fragments were analyzed by agarose gel electrophoresis, stained by ethidium bromide, and visualized under UV illumination.
  • PCR products were purified on a 1 % agarose gel using a Gel extraction kit (Bio Basic Canada Inc., Markham, Ontario, Canada).
  • the rpoB fragments were cloned into a pGEM-T-Easy vectorTM (pGEM-t easy kit, Promega, Medison, USA) and sent for sequencing using the Sp6 and T7 primers (Table 19).
  • the gyrA fragments were sequenced with the same primers that were used for the initial PCR reaction.
  • the gyrB fragments were amplified with the universals UP-1 and UP-2r primers and sequenced with the UP-1 S and the UP-2Sr primers (Table 19).
  • Table 21 Temperature program for amplification of DNA fragments of Bacillus subtilis group specific genes by PCR
  • Table 22 Identification of Bacillus sp. isolates based on sequencing of specific genes
  • B. amyloliquefaciens may produce enzymes and antibiotics, specially bacteriocins, lipopeptides and polyketides.
  • polymerase chain reactions were performed to determine the presence of metabolite genes involved in their biosynthesis. PCR-amplifications were carried out in a 25- ⁇ _ reaction mixture (Table 23) with specific primers for each gene of interest (Table 24) and specific temperature protocol for each amplification reaction (Table 25). After DNA amplification, PCR products were analyzed by agarose gel electrophoresis (1.0% of agarose, 100V, 60 min), DNA was stained by ethidium bromide (0.5 pg/mL), and visualized under UV illumination.
  • Table 24 Amplification primers used to determine the presence of different metabolite genes in Bacillus sp. isolates
  • baeR_F Bacillaene ATGTCAGCTCAGTTTCCGCA 688 bp Compaore et al., 2013 baeR_R GATCGCCGTCTTCAATTGCC
  • Table 25 PCR program for amplification of DNA fragments of genes involved in
  • amylovora 435 B. glumae LMG10905, and P. syringae DC3000
  • B. subtilis ED66 human pathogens
  • S. aureus ED711, P. aeruginosa PA416A human pathogens
  • B. amyloliquefaciens subsp. plantarum 71 inhibited the growth of P. polymyxa To99 and P. polymyxa TFr101 showing a clear halo with diameter 26 mm, while these two strains of P. polymyxa showed the antimicrobial activity against B. amyloliquefaciens subsp. plantarum 71 with 25.16 and 29.00 mm growth inhibitory zones, respectively (Table 28).
  • cell-free supernatant of P. polymyxa To99 contains compounds (e.g., proteinaceous compounds such as bacteriocins or lantibiotics) produced by the bacteria to inhibit the growth of similar or closely related bacterial strains. Metabolites resembling bacteriocins or lantibiotics in structure would likely kill pathogens by binding to lipids on their cell membranes and making the cells porous. This would cause intracellular fluid to leak out, which would destroy the pathogen.
  • Another example of such a molecule is nisin, produced by bacteria found in dairy products.
  • lantibiotics are only effective against Gram-positive bacteria, but they can be induced to destroy Gram-negative pathogens such as those that cause plant diseases, by being combined with a chelator like EDTA. It is clear from above experiments that supernatants produced by these active species have wide-spectrum activity against other bacteria and even against bacteria within the same genus and the same species as the active bacteria.
  • the precultures of P. polymyxa and B. amyloliquefaciens subsp. plantarum isolates were prepared as follows: 5 ⁇ _ of the respective bacterial culture stored at -80°C was inoculated into 3 mL of Tryptic Soy Broth (TSB) and incubated in a rotary shaker at 250 rpm overnight at 30°C. The appropriate aliquots of each culture broth were used to inoculate production media to start with an initial optical density of 0.01 , corresponding to about 8x10 6 CFU/mL.
  • TTB Tryptic Soy Broth
  • Landy's medium contains: glucose 20 g/L, L-glutamic acid 5.0 g/L, yeast extract 1.0 g/L, K 2 HP0 4 1.0 g/L, MgS0 4 (7H 2 0) 0.5 g/L, KCI 0.5 g/L, CuS0 4 1.6 mg/L, Fe 2 (S0 4 ) 3 1.2 mg/L, MnSO 4 0.4 mg/L.
  • the production of metabolites was carried out in 2 L conical flasks with 500 mL of TSB or 200 mL of Landy's medium, and shaken at 250 rpm at 30°C for 48 h. Culture media were then centrifuged at 25 000 rpm for 1 hour. The pellets were discarded and the supernatants were filtered using stericup vacuum filtration system (0.2 ⁇ ). The supernatants were collected and stored at -20°C until usage.
  • campestris 901 using a well-diffusion inhibition assay (Obradovic et al., 2002; WO/2012/051699; Lindow et al., 2003).
  • 10-fold diluted cell-free supernatants of B. amyloliquefaciens subsp. plantarum isolates 71 and VFb49 showed the highest antimicrobial activity amongst the supernatants tested against X. gardneri DC00T7A, X. perforans T4, and X. campestris 901.
  • plantarum isolates 237, 16, and 33 were about 2-fold less active against all thee Xanthomonas species tested, while 10-fold diluted supernatant of B. amyloliquefaciens subsp. plantarum 335 showed no detectable anW-Xanthomas activity in this assay.
  • the five bacterial isolates from Examples 4 and 4.1 that showed the highest activities against Xanthomonas species were used in this example: Bacillus amyliquefaciens subsp. plantarum isolates 71 and VFb49, and Paenibacillus polymyxa isolates To99, 273, and TFr101.
  • Bacillus amyliquefaciens subsp. plantarum isolates 71 and VFb49 Bacillus amyliquefaciens subsp. plantarum isolates 71 and VFb49
  • Paenibacillus polymyxa isolates To99, 273, and TFr101 were used in this example: Bacillus amyliquefaciens subsp. plantarum isolates 71 and VFb49, and Paenibacillus polymyxa isolates To99, 273, and TFr101.
  • the pathogen growth inhibition zone caused by cell-free supernatants of these strains was more than 30 mm in diameter (Table 3, Figure 5), or
  • Bacterial spot of tomato is one of the most pervasive diseases that faces tomato production all over the world. It is caused by four closely related species of Xanthomonas: X. gardneri, X. euvesicatoria, X. vesicatoria, and X. perforans. After a number of early revisions, they were classified for some time as Xanthomonas campestris pv. vesicatoria (Xcv) [Dye, 1978]. Comparative studies of whole genome sequences from reference strains, as well as data of carbon utilization, amylolitic and pectolytic activities of these four species showed considerable diversity between these pathogens, especially the difference between X.
  • Race T1 caused by X. euvesicatoria was the endemic race in Florida until 1991 , when X. perforans race T3 (antagonistic to race T1), emerged.
  • Race T4 came about as a result of a mutation in the X. perforans avrXv3 gene, and has recently become prevalent [Jones et al., 2004; Jones et al., 2005].
  • X. perforans race T4 and X. gardneri DC00T7A were used herein.
  • X. perforans T4 or X. gardneri DC00T7A was incubated in TSB at 30°C for 16h (overnight). Cell culture medium was centrifuged at 10,000xg for 5 min. The supernatant was discarded and bacterial cells were resuspended in sterile distilled water.
  • the NanoDropTM ND-1000 spectrophotometer was set to measure absorbance at 600 nm wavelength, and the suspension was adjusted to an optical density (ODeoonm) of 0.2, which was empirically determined to represent 2x10 8 CFU/mL by plating serial dilutions of the suspension and counting colonies.
  • ODeoonm optical density
  • Tomato Solanum lycopersicum L. var. cerasiforme (Dunal) (D. M. Spooner et al.) cv. Bonny Best (OSC Seeds, Waterloo, Ontario, Canada) or Florida 47 (Harris seeds, Rochester, NY, US) seeds were planted in Pro-mixTM BX MycorrhizaeTM (Premier horticulture Inc., Quakertown, Pennsylvania, USA) with adding all-purpose NPK (20-20- 20) fertilizer (Plant Products Co. LTD., Brampton, Ontario, Canada).
  • Tomato seedlings were grown in plastic pots (6.0x6.0 cm) in a growth chamber (25°C, relative humidity (RH) 40%, with a photoperiod of 16 hours (light intensity is about 200.0 lum/sqf) for two weeks (the four-true-leaf stage) prior to infestation by X. perforans T4 or . gardneri DC00T7A.
  • Temperature, relative humidity (RH), and lightness were monitored hourly during the research using HoboTM digital system (Onset computer corporation) that was located in the center of the shelf with tomatoes.
  • the trials under growth chamber conditions were carried out on tomato seedlings to assess the efficacy of cell-free bacterial supernatants of B. amyliquefaciens subsp. plantarum and P. polymyxa isolates in controlling artificial infections caused by X. perforans T4, as compared to a mix of copper and mancozeb (2 and 1 g/L, respectively).
  • Tomato seedlings were sprayed on abaxial and adaxial leaf surfaces to leaf wetness with a hand sprayer, applying approximately 2 ml_ of X. perforans T4 suspension per plant. After 1 h of inoculation, seedlings were treated by spraying cell-free supernatants, water, or TSB, respectively, using a hand-pump up to run-off of plants. Tomato seedlings were maintained for 48h under plastic bags and then placed in a growth chamber (30°C, RH 60% with a photoperiod of 16 hours) for one week.
  • the plants were assessed for disease severity by visual estimation of the percentage of leaf tissue with spots and lesions 10 days after infestation. Symptoms on the leaves were recorded for each plant by two persons independently. Disease severity assessments were made based on leaf rating compiled from three separate experiments.
  • P. polymyxa and B. amyloliquefaciens subsp. plantarum isolates were removed from frozen stock vials and 5 ⁇ _ of each isolate was added to 3 ml_ of TSB.
  • Foliar treatment of tomato seedlings was performed by suspension of live bacterial cells (3 x 10 8 CFU/mL) of Bacillus and Paenibacillus isolates, and respective control solutions using a hand trigger sprayer with application on abaxial and adaxial leaf surfaces until run-off.
  • live bacterial cells 3 x 10 8 CFU/mL
  • paenibacillus isolates 3 x 10 8 CFU/mL
  • respective control solutions using a hand trigger sprayer with application on abaxial and adaxial leaf surfaces until run-off.
  • leaf print on Tryptic Soy Agar (TSA) plates and serial dilution method were performed 1 hour and 6 days after treatment.
  • Figure 12 shows a typical print of an untreated tomato leaf in panel (A), and one treated with B. amyloliquefaciens subsp. plantarum 71 panel (B) after 24 hours of incubation.
  • FIG. 13 shows a whitish film on tomato leaves formed by Bacillus and Paenibacillus isolates 6 days after respective treatments (panels A, C, E, G, I), as well as leaf shape of their live bacterial colonies after leaf printing (panels B, D, F, H, J).
  • Bacterial cells of all Bacillus and Paenibacillus isolates tested in this Example were still alive on tomato leaves even 6 days following treatments, but the quantity of CFUs per leaf was different depending on the isolates.
  • 1 hour after treatment the leaves treated by B. amyloliquefaciens subsp. plantarum 71 contained the highest quantity (log of CFU/leaf reached 3.77) of bacterial live cells, while the quantity of P. polymyxa 273 live cells was 1.6-fold less (log of CFU/leaf reached up 2.33).
  • the quantity of CFU/leaf of Bacillus and Paenibacillus isolates increased by about 1.5 fold.
  • Tomato seedlings were sprayed on abaxial and adaxial leaf surfaces to leaf wetness with a hand sprayer, applying approximately 2 ml_ of cell-free bacterial supernatants or live cell suspension per plant. Water and TSB were used as controls. After treatment, plants were placed in a growth chamber (30°C, RH 60% with a photoperiod of 16 hours) for one week. Then, tomato seedlings were infected by X. gardneri DC00T7A in the same way using a hand- pump up to run-off of plants and placed in a growth chamber. After 10 days, the effectiveness of cell-free supernatants and live cells was evaluated as the reduction of spot numbers per plant compared to the pathogen-only control. The results were compiled from three separate experiments.
  • the cell-free supernatants exhibiting the highest resistance to light exposure were those from the tested strains of Paenibacillus polymyxa, whose activity began to decrease only after 3 weeks of light exposure.
  • the P. polymyxa-demed supernatants exhibited activity against X. gardneri DC00T7A even after 12 weeks of light exposure, in contrast to those from the B. amyloliquefaciens subsp. plantarum strains.
  • P. polymyxa To99 exhibited the highest activity against X. gardneri DC00T7A.
  • control is the activity of fresh supernatants after 1 h storage at 4°C and 22°C, respectively.
  • the dried sample was dissolved in 2 ml_ of dhbO.
  • the antimicrobial activity of undiluted and 10-fold diluted fractions was estimated using well diffusion method by measuring the inhibition area (mm 2 ) of Xanthomonas gardneri DC00T7 A, as described in Example 6.1. The results are shown in Table 31.
  • the anti-Xanthomonas activity of organic and aqueous phases of all 5 bacterial isolates indicates that they produce several compounds, including lipopeptides, with different diffusion abilities.
  • 100% organic phase (lipopeptides) of P. polymyxa To99 supernatant showed the same anW-Xanthomonas activity as 100% crude supernatant, while 100% aqueous phase of its supernatant was 1.2-fold more active against X. gardneri DC00T7A.
  • the aqueous phases of their supernatants were more active against X. gardneri DC00T7A than their organic phases.
  • the whole genome sequencing was performed to further confirm the taxonomic identification of the isolates, in addition to the results already obtained from the sequencing of 16S rDNA (Example 2.2) and specific ⁇ gyrA, gyrB and rpoB) genes (Examples 2.3 and 2.5), biochemical tests of BiologTM microbial identification system (Example 2.3a), and fatty acid analyses (Example 2.4).
  • Genomic DNA was isolated from an overnight culture of each strain (71 , VFb49, To99, TFr101 , and 273) using a Qiagen DNeasyTM blood and tissue kit (Qiagen Inc., Valencia, CA). Genome sequencing was performed using lllumina MiSeqTM sequencing system (lllumina, San Diego, CA), achieving >50x average genome coverage. De novo assembly was created for each genome using SPAdesTM 3.0.0 (St. Orlando genome assembler), and annotated with the NCBI Prokaryotic Genomes Automatic Annotation Pipeline (http://www.ncbi.nlm.nih .gov/genomes/static/Pipeline.html).
  • Taxonomy of each strain was assigned using KrakenTM (Wood and Salzberg, 2014), a metagenomics sequence classification tool (http://ccb.jhu.edu/software/kraken/). Genome mining of biosynthetic gene clusters, including non-ribosomal peptide synthetases (NRPSs) and other secondary metabolites, were predicted with antibiotics & Secondary Metabolite Analysis SHellTM (antiSMASHTM) (Weber et al., 2015) web server (http://antismash.secondarymetabolites.org/). 7.2 Results
  • the ANI calculator was used (http://enve-omics.ce.gatech.edu/ani/index) to compare the nucleotide identity of the bacterial isolates with already known and type strains of Paenibacillus and Bacillus species from the NCBI database (http://www.ncbi.nlm.nih.gov/nuccore).
  • the ANI calculator estimates the average nucleotide identity using both best hits (one-way ANI) and reciprocal best hits (two-way ANI) between two genomic datasets, as calculated by Goris et al., 2007. Typically, the ANI values between genomes of the same species are above 95%. Table 32.2.
  • the average nucleotide identity (%) based on complete genome sequencing between Bacillus (A) and Paenibacillus (B) isolates possessing anW-Xanthomonas activity and type strains
  • VFb49 and 71 isolates showed about 99% nucleotide identity with Bacillus amyloliquefaciens subsp. plantarum (Table 32.2A), which confirms our previous identification based on sequencing of gyrA, gyrB and rpoB genes, specific for Bacillus subtilis group.
  • the antibiotics Secondary Metabolite Analysis Shell (antiSMASHTM) tool allowed us to identify 3 polyketide synthetases (PKSs), 15 non-ribosomal peptide synthetases (NRPSs), and 3 hybrid PKSs/NRPSs genes that could be involved in the synthesis of secondary metabolites of Bacillus amyloliquefaciens subsp. plantarum and Paenibacillus polymyxa isolates possessing the highest anW-Xanthomonas activity (Table 32.3). Genome of S. amyloliquefaciens subsp. plantarum (isolates 71 and VFb49) and P.
  • polymyxa (isolates To99, TFr101 and 273) harboured different gene clusters responsible for the production of polyketides, lipopeptides, bacteriocins and lantibiotics putatively involved in controlling bacterial leaf spot caused by Xanthomonas species (Table 32.3).
  • Table 32.3 Presence of genes belonging to biosynthetic clusters encoding secondary metabolites of Bacillus
  • B. amyloliquefaciens subsp. plantarum 71 resulted in the identification of gene clusters for the synthesis of polyketides such as bacillaene, difficidin, macrolactin.
  • B. amyloliquefaciens subsp. plantarum VFb49 does not possess gene cluster encoding macrolactin production, while bacillaene and difficidin gene clusters were revealed in its genome.
  • gene clusters involved in polyketide production by P. polymyxa isolates the genes clusters responsible for the synthesis of bacillaene and nosperin were revealed in P. polymyxa isolates To99 and 273.
  • the genes encoding kalimantacin/batumin and myxovirescin were discovered in B. amyloliquefaciens subsp. plantarum 71 and P. polymyxa 273 genomes (Table 32.3).
  • polymyxa possess gene cluster responsible for the synthesis of NRPs such as polymyxin and fusaricidin. Gene clusters involved in bacillomycin and pelgipeptin biosynthesis were found in P. polymyxa isolates To99 and TFr101. Gene cluster responsible for tridecaptin and bacitracin synthesis was discovered in the genome of P. polymyxa isolates TFr101 and 273. Only the genome of P. polymyxa To99 possesses gene clusters involved in synthesis of paenilarvin and paenibacterin..
  • Gene clusters responsible for the peptide biosynthesis by RPSs such as plantathiazolicin/plantazolicin and bacteriocins were detected only in the genome of B. amyloliquefaciens subsp. plantarum 71.
  • B. amyloliquefaciens subsp. plantarum VFb49 possesses gene cluster for subtilin, while gene clusters encoding paenicidin B and paenibacillin biosynthesis are found in the genome of P. po/ymyxa TFr101 (Table 32.3).
  • Xanthomonas activity harbour gene clusters involved in the biosythensis of cyclic lipopeptides such as surfactin, fengycin, plipastatin and iturin; as well as of the siderophore, bacillibactin.
  • the isolate VFb49 also harbour gene clusters involved in the biosythensis of locillomycin, a new family of cyclic lipopeptides (Luo et al., 2015).
  • the copper fungicide (KocideTM 3000) was used at 1.68 kg/ha + Tanos at 0.42 kg/ha with 200 L/ha water volume. Initial treatments were applied 2 DBI as foliar spray, followed by foliar spray applications every 7 days +/- 1 day for 4 more weeks for a total of 5 applications.

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Abstract

Anti-microbial metabolites secreted from various bacterial and/or fungal species are described that have anti-bacterial activity against Xanthomonas species, which cause disease in plant hosts, including a wide variety of crops. Bacterial and fungal species producing such metabolites were identified from environmental samples (seeds, different vegetable organs and soil) from different geographic locations. Compositions, methods, uses, and kits 5 relating to the identified anti-microbial metabolites are also described.

Description

BACTERIAL AND FUNGAL METABOLITES POSSESSING ANTI-MICROBIAL ACTIVITY AGAINST XANTHOMONAS SPECIES, COMPOSITIONS, METHODS, KITS AND USES RELATING TO SAME
The present invention relates to anti-microbial metabolites from various bacterial and fungal species. More particularly, the present invention relates to bacterial and fungal metabolites having anti-microbial activity against Xanthomonas species.
BACKGROUND
Xanthomonas is a genus of Gram-negative rod-shaped bacteria that acts as a plant pathogen. Many Xanthomonas species cause serious diseases in hundreds of plant hosts, including a wide variety of economically important crops, such as tomato, pepper, lettuce, strawberries, walnuts, rice, citrus, banana, cabbage, and bean.
Approaches to control Xanthomonas pathogenic species on plants include the use of non-specific chemicals such as copper-based formulations, in which copper ions bind indescriminantly to sulfhydryl groups, accounting for their non-specific biocidal and anti-bacterial activity. However, free copper ions can penetrate through plant cuticles and cause severe phytotoxicity. Furthermore, reduced copper sensitivity among Xanthomonas strains has been reported in some areas
(i.e., some Xanthomonas strains became copper-tolerant), necessitating the addition of other agents such as Maneb™ or Mancozeb™ fungicides to the copper-based formulations to increase efficacy. The use of copper-based formulations may also have a negative environmental impact, since copper ions are not degraded in soil and can accumulate to high levels at locations with a history of intensive copper application.
Alternative chemical control approaches have been investigated in which chemicals are applied that activate plant defense responses. For example, Systemic Acquired Resistance (SAR) is a biochemical state of the plant in which the plant develops greater resistance to a pathogen by previous infection by that pathogen or a different pathogen. Several substances that induce SAR have been investigated (e.g., acibenzolar-S-methyl; ASM). However, such SAR inducers may reduce crop yield since it is argued that energy is spent to activate the plant defense system instead of growth.
Biological control of plant diseases may offer a safer and more specific effective alternative to the use of synthetic chemicals, and may pose less environmental concerns. There is thus a need for biological products for controlling plant pest diseases, such as Xanthomonas species. SUMMARY
The present inventors have unexpectedly discovered anti-microbial metabolites secreted by various bacterial and/or fungal species that have anti-bacterial activity against Xanthomonas species, which cause disease in plant hosts, including a wide variety of crops. Bacterial and fungal species producing these metabolites were identified from environmental samples (seeds, different vegetable organs and soil) from different geographic locations. Compositions, methods, uses, and kits relating to the identified anti-microbial metabolites are also described herein.
Accordingly, in some aspects, the present description relates to a composition comprising metabolites from a bacterial and/or fungal species, wherein the metabolites have antimicrobial activity against Xanthomonas species. In some embodiments, the metabolites may be extracellular bacterial and/or extracellular fungal metabolites (e.g., secondary metabolites). In some embodiments, the metabolites may be from a: Bacillus species; Paenibacillus species; Burkholderia species; Mortierella species; Giberella species; Fusarium species; Aspergillus species; Penicillium species; or any combination thereof. In some embodiments, the metabolites may be from: Paenibacillus polymyxa; Paenibacillus peoriae; Bacillus amyloliquefaciens; Burkholderia cepacia; Mortierella alpine; Giberella moniliformis; Fusarium oxysporum; Aspergillus n/ger Tiegh; Aspergillus hiratsukae; Penicillium ochrochloron; or any combination thereof. In some embodiments, the metabolites may be from Burkholderia cepacia, Paenibacillus polymyxa, Paenibacillus peoriae, and/or Bacillus amyloliquefaciens. In some embodiments, the metabolites may be from Paenibacillus peoriae. In some embodiments, the metabolites may be from Bacillus amyloliquefaciens. In some embodiments, the metabolites may be from Paenibacillus polymyxa T1 B; Paenibacillus polymyxa 44; Paenibacillus sp. 62; Paenibacillus polymyxa 273 (since renamed as Paenibacillus peoriae 273); Paenibacillus polymyxa 329; Paenibacillus sp. 344; Paenibacillus polymyxa 390; Paenibacillus polymyxa To99 (since renamed as Paenibacillus peoriae To99; deposited on March 9, 2015 at the NRRL under No NRRL B-67020); Paenibacillus polymyxa TP12; Paenibacillus polymyxa TP29; Paenibacillus polymyxa TP77; Paenibacillus polymyxa V25T; Paenibacillus polymyxa TFr60; Paenibacillus sp. TFr101 (since renamed as Paenibacillus peoriae TFr101 ; deposited on March 9, 2015 at the NRRL under No NRRL B-67019); Paenibacillus polymyxa TAu1 ; Paenibacillus polymyxa TM54; Bacillus amyloliquefaciens subsp. plantarum 16; Bacillus amyloliquefaciens subsp. plantarum 33; Bacillus amyloliquefaciens subsp. plantarum 71 (deposited on March 9, 2015 at the NRRL under No NRRL B-67021); Bacillus amyloliquefaciens subsp. plantarum 237; Bacillus amyloliquefaciens subsp. plantarum 335; Bacillus amyloliquefaciens subsp. plantarum VFb49; Burkholderia cepacia BC19; Burkholderia cepacia BC153; Mortierella sp. VFb1 ; Giberella sp. TFr4; Fusarium sp. FI3S; Aspergillus sp. 8PT; Aspergillus sp. FG; Penicillium sp. VFr37; or any combination thereof. In some embodiments, the metabolites may be from: Bacillus amyloliquefaciens subsp. plantarum 71 (deposited on March 9, 2015 at the NRRL under No NRRL B-67021); Paenibacillus polymyxa To99 (since renamed as Paenibacillus peoriae To99; deposited on March 9, 2015 at the NRRL under No NRRL B-67020); Paenibacillus polymyxa TFr101 (since renamed as Paenibacillus peoriae TFM01 ; deposited on March 9, 2015 at the NRRL under No NRRL B-67019); or any combination thereof. In some embodiments, the metabolites may have antimicrobial activity against phytopathogenic Xanthomonas species. In some embodiments, the metabolites may have antimicrobial activity against Xanthomonas campestris, Xanthomonas perforans, Xanthomonas gardneri, or any combination thereof. In some embodiments, the Xanthomonas campestris may comprise Xanthomonas campestris MAPAQ #901 and/or Xanthomonas campestris ED1985, or the Xanthomonas gardneri may comprise Xanthomonas gardneri DC00T7A. In some embodiments, the Xanthomonas perforans may comprises Xanthomonas perforans T1 , T2, T3, T4, T5, or any combination thereof. In some embodiments, the Xanthomonas perforans may comprise Xanthomonas perforans T4. In some embodiments, the metabolites may further have antimicrobial activity against a plant and/or a human pathogenic microorganism. In some embodiments, the pathogenic microorganism may be a virus, bacteria, fungus (including a microscopic fungus), yeast, mold, or any combination thereof. In some embodiments, the pathogenic microorganism may be: Xanthomonas euvesicatoria; Xanthomonas fraganae; Xanthomonas perforans; Xanthomonas campestns; Xanthomonas gardneri; Pseudomonas syringae; Erwinia amylovora; Burkholderia glumae; Escherichia coli; Bacillus subtilis; Staphylococcus aureus; Pseudomonas aeruginosa; or any combination thereof. In some embodiments, the pathogenic microorganism may be: Xanthomonas euvesicatoria R4; Xanthomonas gardneri DC00T7A; Xanthomonas fraganae LMG 708; Pseudomonas syringae DC3000; Erwinia amylovora 435; Burkholderia glumae LMG10905; Escherichia coli 0157:H7 EDL933; Bacillus subtilis ED66; Staphylococcus aureus ED711; Pseudomonas aeruginosa PA416A; or any combination thereof. In some embodiments, the antimicrobial activity may comprise anti-bacterial and/or anti-fungal antagonistic activity. In some embodiments, the metabolites may exhibit higher antimicrobial activity against Xanthomonas species, as compared to other species. In some embodiments, the metabolites may comprise lipopeptides and/or siderophores having anW-Xanthomonas activity. In some embodiments, the lipopeptides and/or siderophores may be from Bacillus and/or Paenibacillus. In some embodiments, the lipopeptides may be non-ribosomal lipopeptides (NRPs). In some embodiments, the lipopeptides and/or siderophores may comprise surfactin, fengycin, plipastatin, iturin, bacilysin, bacillibactin, bacillomycin, locillomycin, paenilarvin, pelgipeptin, polymyxin, paenibacterin, fusaricidin, bacitracin, tridecaptin, or any combination thereof. In some embodiments, the metabolites may comprise plipastatin and/or locillomycin. In some embodiments, the composition defined herein may further comprise an agriculturally acceptable excipient. In some embodiments, the composition defined herein may further comprise one or more of: non-toxic carriers, surfactants, preservatives, nutrients, UV protectants, stickers, spreaders and chelating agents. In some embodiments, the composition may lack viable cells from the bacterial and/or fungal species. In some embodiments, the composition may be a cell-free composition. In some embodiments, the composition may be a cell- free supernatant. In some embodiments, the composition may comprise killed cells from the bacterial and/or fungal species. In some embodiments, the composition may lack viable cells from the bacterial and/or fungal species. In some embodiments, the composition may comprise viable cells from the bacterial and/or fungal species. In some embodiments, the composition may comprise spores from the bacterial and/or fungal species. In some embodiments, the composition may be in the form of a liquid, concentrate, powder, tablet, gel, pellets, granules, or any combination thereof. In some embodiments, the composition, once applied to a target plant, may have no detectable phytotoxic effect on the target plant, or on the fruits, nuts, or leaves thereof. In some embodiments, the composition may comprise at least 100 ppm, 200 ppm, 300 ppm, 400 ppm, 500 ppm, 600 ppm, 700 ppm, 800 ppm, 900 ppm, 1000 ppm, 1500 ppm, 2000 ppm, 2500 ppm, 3000 ppm, 3500 ppm, 4000 ppm, 4500 ppm, 5000 ppm, 5500 ppm, 6000 ppm, 6500 ppm, 7000 ppm, 8000 ppm, 8500 ppm, 9000 ppm, or 9500 ppm of the metabolites. In some embodiments, the composition may comprise between about 100 ppm, 200 ppm, 300 ppm, 400 ppm, 500 ppm, 600 ppm, 700 ppm, 800 ppm, 900 ppm, 1000 ppm, 1500 ppm, 2000 ppm, 2500 ppm, 3000 ppm, 3500 ppm, 4000 ppm, 4500 ppm, 5000 ppm, to about 10 000 ppm of the metabolites. In some embodiments, the compositions defined herein may be for use as an anti-microbial agent against a plant and/or human pathogenic microorganism. In some embodiments, the composition as defined herein may be a biopesticide.
In some aspects, the present description relates to the use of the compositions as defined herein as an antimicrobial agent against a plant and/or human pathogenic microorganism. In some aspects, the present description relates to the use of the compositions as defined herein for the manufacture of an anti-microbial agent against a plant and/or human pathogenic microorganism. In some embodiments, the pathogenic microorganism may be a Xanthomonas species. In some embodiments, the compositions or uses defined herein may be for application to a growing plant. In some embodiments, the growing plant may be a fruit plant, nut, cereal, vegetable, or flower. In some embodiments, the fruit may be: apple, apricot, banana, blackberry, blueberry, cantaloupe, cherry, cranberry, currant, grapes, greengage, gooseberry, honeydew, lemon, mandarin, melon, orange, peach, pears, pineapple, plum, raspberry, strawberry, tomatoes, watermelon, grapefruit, pepper, olive, or lime. In some embodiments, the nut may be: almond, beech nut, Brazil nut, butternut, cashew, chestnut, chinquapin, filbert, hickory nut, macadamia nut, pecan, walnut, or pistachio. In some embodiments, the cereal may be: amaranth, breadnut, barley, buckwheat, canola, corn, fonio, kamut, millet, oats, quinoa, cattail, chia, flax, kahiwa, pitseed goosefoot, wattleseed, rice, rye, sorghum, spelt, teff, triticale, wheat, or colza. In some embodiments, the vegetable may be: artichoke, bean, beetroot, broad bean, broccoli, cabbage, carrot, cauliflower, celery, chicory, chives, cress, cucumber, kale, dill, eggplant, kohlrabi, lettuce, onion, pepper, parsnip, parsley, pea, potato, pumpkin, radish, shallot, soybean, spinach, turnip, or peanut. In some embodiments, the growing plant may be a tomato plant; a pepper plant; a berry plant; a strawberry plant; lettuce; a citrus plant; a walnut plant; a rice plant; and/or a kiwi plant. In some embodiments, the flower may be: a species of the Euphorbiaceae; Euphorbia pulcherrima (poinsettia), Euphorbia milii (crown-of- thorns), Codiaeum variegatum (croton); a member of the family Rosaceae (Rosoideae/Rosa), Begoniaceae (Begonia), Araceae; Dieffenbachia, Anthurium, Philodendreae (Philodendron), Caladieae (Syngonium), English ivy or another Araliaceae species; Pelargonium (geranium), Ficus, Hydrangea, Zinnia, ornamental Prunus species, ornamental Peppers, or another flower and/or ornamental plant susceptible to infection by Xanthamonas. In some embodiments, the compositions or uses defined herein may be for application to a plant cell or tissue which may be: a leaf, a stem, a flower, a fruit, a tuber, a rhizome, a corm, a root, or any combination thereof.
In some aspects, the present description relates to a method for producing a composition as defined herein, the method comprising culturing viable cells from the bacterial and/or fungal species to produce the metabolites; and harvesting the metabolites produced therefrom. In some embodiments, the viable cells may be cultured in Landy medium or Tryptic Soy Broth (TSB). In some aspects, the present description relates to an antimicrobial composition produced by a method defined herein. In some aspects, the present description relates to a method for controlling the growth of a pathogenic microorganism on a target plant or tissue; the method comprising contacting the target plant or tissue with a composition as defined herein. In some embodiments, the contacting comprising spraying, irrigating, painting, daubing, and/or fogging, onto and/or into the target plant or tissue, the target plant or tissue's hydroponic substrate, and/or the target plant or tissue's agricultural earth.
In some aspects, the present description relates to a kit for preparing an aqueous solution for use in controlling pathogens on a plant tissue of a growing plant, the kit comprising: (a) an organism as defined herein which produces metabolites having antimicrobial activity against Xanthomonas species, and/or a composition as defined herein; and (b) a suitable container. In some embodiments, the container may be a pouch, a tablet, or a bucket. In some embodiments, the kit may be for use in controlling infection by the pathogenic microorganism on a plant. In some embodiments, the pathogenic microorganism may be ^ Xanthomonas species.
DEPOSIT OF BIOLOGICAL MATERIAL
Purified cultures of each of the bacterial strains Bacillus amyloliquefaciens subsp. plantarum 71 ; Paenibacillus peoriae To99; and Paenibacillus peoriae TFr101 were deposited at the Agricultural Research Service Culture Collection (NRRL) (USDA, ARS, 1815 North University Street, Peoria, III, 61604, USA) on March 9, 2015. The deposits were made under the terms of the Budapest Treaty. "Bacillus amyloliquefaciens subsp. plantarum 71" has been assigned Accession number NRRL B-67021 ; "Paenibacillus peoriae To99" has been assigned Accession number NRRL B-67020; and "Paenibacillus peoriae TFr101" has been assigned Accession number NRRL B-67019. At the time of the deposit, isolates To99 and TFr101 were initially identified as "Paenibacillus polymyxa". Further whole genome sequencing analyses presented herein (Example 7) subsequently revealed that these isolates may be more accurately classified as "Paenibacillus peoriae". Thus, in some sections of the present description, while the isolates To99 and/or TFr101 may be still be referred to as Paenibacillus polymyxa, it is understood that they may also be referred to as Paenibacillus peoriae, while still referring to the same deposited organisms.
BRIEF DESCRIPTION OF THE DRAWINGS
In the appended drawings:
Figure 1 shows the results of a phylogenetic analysis of bacteria within the genus Xanthomonas and the related genera Xylella and Stenotrophomonas.
Figure 2 shows an example of how the isolated bacterial strains were screened to identify those having antagonistic activity against X. campestris ED740 (A) and X. campestris ED1985 (B). Halo boundaries are delineated by dotted circles.
Figure 3 shows antagonistic activity of the bacterial strain "To65" against X. campestris ED1985. The halo boundary around the bacterial colony of strain "To65" is indicated with a dotted circle. Figure 4 shows an example of how isolated fungal strains were screened to identify those having antagonistic activity against X. campestris ED1985. The halo boundary around the fungal colony of strain "TAu20" is indicated with a dotted circle.
Figure 5 shows the antimicrobial activity of three bacterial strains against X. perforans T4: Bacillus amyloliquefaciens subsp. plantarum 71 ("71"); P. polymyxa TFr101 ("TFr101"); and P. polymyxa To99 ("To99"). Halo boundaries are delineated by dotted circles.
Figure 6 shows the antimicrobial activity of several fungal strains against X. perforans T4: (A) Penicillium sp. VFr37 ("VRf37"); and (B) Giberella sp. TFr4 ("TFr4"), Fusarium sp. FI3S ("FI3S"). Halo boundaries are delineated by dotted circles.
Figure 7 shows the agarose gel electrophoresis profiles of a PCR-amplified 390-bp region of the recA gene in two bacterial isolates: "19" and "153". "+" is Burkholderia cepacia ATCC 25416, "-" is a negative control. Molecular sizes in base pairs of the DNA standard (GeneRuler™ 1 Kb DNA Ladder, Fermentas) are indicated in the left-hand margin.
Figure 8 shows the bactericidal effect of a solution of coppenmancozeb (2:1) against Xanthomonas perforans T4. The halo boundary is indicated with a dotted circle.
Figure 9 presents photographs showing the effects of different treatments on leaf spots on the leaves of tomato seedlings. In the individual panels, A: not-infected and treated by water; B: infected by X. perforans T4 and treated by Tryptic Soy Broth (TSB); C & D: infected by X. perforans T4 and treated by water - upper (C) and lower (D) leaves surfaces are shown; E & F: infected by X. perforans T4 and treated by copper plus mancozeb - upper (E) and lower (F) leaves surfaces are shown; G & H: infected by X. perforans T4 and treated by cell-free supernatant of B. amyloliquefaciens subsp. plantarum 71 - upper (G) and lower (H) leaves surfaces are shown; I & J: infected by X. perforans T4 and treated by cell-free supernatant of P. polymyxa To99 - upper (I) and lower (J) leaves surfaces are shown; K & L: infected by X. perforans T4 and treated by cell-free supernatant of P. polymyxa TFr101 : upper (K) and lower (L) leaves surfaces are shown; M: not-infected and treated by cell-free supernatant of B. amyloliquefaciens subsp. plantarum 71 ; and N: not-infected and treated by cell-free supernatant of P. polymyxa To99; 0 & P: infected by X. perforans T4 and treated with cell-free supernatant of B. amyloliquefaciens subsp. plantarum VFb49: adaxial (0) and abaxial (P) leaves surfaces are shown; Q & R: infected by X. perforans T4, treated with cell-free supernatant of P. polymyxa 273: adaxial (Q) and abaxial (R) leaves surfaces.
Figure 10 shows the antimicrobial activity of Bacillus amyloliquefaciens subsp. plantarum 10-fold diluted cell-free supernatants against Xanthomonas species.
Figure 11 shows the antimicrobial activity of Paenibacillus polymyxa 10-fold diluted cell-free supernatants against Xanthomonas species.
Figure 12 shows a typical print of an untreated tomato leaf in panel (A), and one treated with B. amyloliquefaciens subsp. plantarum 71 in panel (B) after 24 hours of incubation. Figure 13 shows microbial viability of bacterial isolates on tomato leaves as evaluated by the leaf print method. A whitish film on tomato leaves formed by Bacillus and Paenibacillus isolates 6 days after respective treatments (panels A, C, E, G, I), as well as leaf shape of their live bacterial colonies after leaf printing (panels B, D, F, H, J) are shown.
Figure 14 shows the quantity of CFUs per leaf at 1 h or 6 h following treatment of tomato leaves with live bacterial cells of the indicated Bacillus and Paenibacillus isolates, as compared to untreated tomato leaves.
Figure 15 shows the colonies formed on TSA by microorganisms isolated from untreated tomato leaves after 6 days (panel C), or from tomato leaves treated 6 days prior with B. amyloliquefaciens subsp. plantarum 71 (panel A) or P. polymyxa To99 (Panel B).
Figure 16 shows yellow-brownish lesions caused by X. perforans T4 on tomato leaves, susceptible reaction caused by X. gardneri DC00T7A manifested 10 days after infestation as well-defined brown spots appeared on leaves (panels A and B) and stems (panels C and D). Brown spot are indicated with arrows.
Figure 17 shows the preventative effect of pre-treating the leaves of tomato seedlings with live cells of Bacillus and Paenibacillus isolates, or their metabolites (supernatants), followed by infection with X. gardneri DC00T7A. Results are expressed as the quantity of spots per plant.
Figure 18 shows the results of agar disc diffusion assays performed in order to evaluate the sensitivity of bacterial metabolites (cell-free supernatants) to light exposure for 0 to 12 weeks. Antimicrobial activity was then tested weekly by placing the paper discs saturated with 10-fold diluted cell-free supernatants on a lawn of Xanthomonas gardneri DC00T7A and measuring the inhibition area (in mm2). (A) B. amyloliquefaciens subsp. plantarum 71 and VFb49; (B) P. polymyxa To99, TFr101 , and 273.
Figure 19 shows the antimicrobial activities of Paenibacillus and Bacillus 10-fold diluted cell-free supernatants against X. gardneri DC00T7A after storage at -20°C for 0, 6 or 12 months.
Figures 20-23 show the results of four greenhouse trials that were conducted to determine the efficacy of metabolites and/or live bacterial strains to control bacterial leaf spots, as compared to standard chemicals agents.
DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
In some aspects, the present invention relates to a composition comprising metabolites from a bacterial and/or fungal species, wherein said metabolites have antimicrobial (e.g., bactericidal) activity against Xanthomonas species.
The use of the word "a" or "an" when used in conjunction with the term "comprising" in the claims and/or the specification may mean "one" but it is also consistent with the meaning of "one or more", "at least one", and "one or more than one".
As used herein, the term "metabolites" refers to any compound, substance or by-product obtainable by the culture or fermentation of a microorganism as described herein. In some embodiments, the metabolites of the present description may be produced by culturing microorganisms and harvesting extracellular metabolites produced therefrom (e.g., released into the culture supernatants). In other embodiments, the metabolites of the present description may be produced using recombinant DNA technology (e.g., recombinant proteins). In some embodiments, the metabolite may be a proteinaceous substance (i.e., a substance comprising a linear polymer chain of at least 3 amino acids bonded together by peptide bonds), bacteriocins, lantibiotics, lipopeptides and/or polyketides. In some embodiments, the metabolites may be extracellular bacterial and/or extracellular fungal secondary metabolites. As used herein, "secondary metabolites" refers to compounds that are not directly involved in normal growth, development, or reproduction. Unlike primary metabolites, the absence of secondary metabolites does not result in immediate death, but rather in long-term impairment of survivability, fecundity or aesthetics.
As used herein, the expression "antimicrobial" refers to the ability of the metabolites of the present description to prevent, inhibit, and/or destroy the growth of pathogenic microbes such as pathogenic bacteria and/or pathogenic fungi. In some embodiments, the expression "antimicrobial" encompasses agents or compounds exhibiting antagonistic activity against pathogenic microbes. In some embodiments, the antimicrobial activity may be in vitro antimicrobial activity or in vivo antimicrobial activity.
In some embodiments, the present description relates to metabolites having antimicrobial activity against phytopathogenic Xanthomonas species. As used herein, term "pathogen" or "pathogenic" refers to an organism capable of producing a disease in a plant or animal. The term "phytopathogen" as used herein refers to a pathogenic organism that infects a plant.
As used herein, the expression "Xanthomonas species" refers to a microorganism belonging to the genus Xanthomonas (including pathogenic Xanthomonas species). Phylogenetic analysis of bacteria within the genus Xanthomonas and the related genera Xylella and Stenotrophomonas is shown in Figure 1. In some embodiments, "Xanthomonas species" includes the Xanthomonas species listed in Figure 1. Without being bound by theory, the virulence of Xanthomonas species may be due to their secretion of extracellular enzymes such as endoglucanases (e.g., carboxymethylcellulases) that hydrolyze cellulose. Xanthomonas species produce also high molecular weight exopolysaccharides (EPS) xanthan and effectors of type III secretion that contribute to its virulence (Ray et al., 2000).
In some embodiments, the metabolites of the present description may have antimicrobial activity against Xanthomonas campestris and/or Xanthomonas perforans. In more particular embodiments, the Xanthomonas campestris may comprise Xanthomonas campestris MAPAQ #901 and/or Xanthomonas campestris ED 1985.
There are at least five races of Xanthomonas spp. (T1 , T2, T3, T4, and T5), which were described as causal agents of bacterial spot in tomato. Jones et al. (2004) proposed a new classification for the genus, as follows: race T1 was identified as X. euvesicatoria; race T2 as X. vesicatoria, and the races T3, T4 and T5 were identified as Xanthomonas perforans (Jones et al., 2004). Race T4 came about as a result of a mutation in the X. perforans avrXv3 gene, and has become prevalent and a major problem on tomato in the state of Florida (USA). Accordingly, in some embodiments, the metabolites of the present description may have antimicrobial activity against Xanthomonas perforans T1 , T2, T3, T4, T5, or any combination thereof. In a more particular embodiment, the metabolites of the present description may have antimicrobial activity against Xanthomonas perforans T4. In some instances, new phylogenic analyses of bacterial and/or fungal species (and others) have resulted in taxonomic reclassifications. Such changes in taxonomic classification are within the scope of the present invention and, regardless of future reclassifications, a person of skill in the art would be able to identify the organisms of the present description using methods described herein and other methods within the capabilities of the skilled person.
In some embodiments, the present description relates to metabolites which may be extracellular bacterial and/or extracellular fungal metabolites. As used herein, the term "extracellular" refers to the compounds that are secreted or released (either actively or passively) into the extracellular medium upon culture of viable cells, but may also include compounds that contact the extracellular medium, but which remain associated with the cell membrane.
In some embodiments, the present description relates to metabolites from a: Bacillus species; Paenibacillus species; Burkholderia species; Mortierella species; Giberella species; Fusarium species; Aspergillus species; Penicillium species; or any combination thereof. As use herein, the expression "from a [genus] species" or "obtainable from a [genus] species", refers to a compound that may be obtained (i.e., that is obtainable) from the culture or fermentation of a species belonging to the recited genus, but does not necessarily mean that the metabolite must be obtained from that particular species or from the culture of a microorganism perse. For example, compounds produced recombinantly or synthetically, but which have a structure substantially corresponding to the metabolite from the recited species, are also encompassed in the aforementioned expressions. In contrast, as used herein, the expression "produced from" is intended to refer to a compound which is obtained from the culture or fermentation of a microorganism of the present description.
In some embodiments, the present description relates to metabolites from an organism that is naturally- occuring and/or that has not been genetically modified using recombinant DNA technology, and thus qualifies as a natural biopesticide and/or natural bioproduct.
In some embodiments, the present description relates to metabolites are from a: Bacillus species; Paenibacillus species; Burkholderia species; Mortierella species; Giberella species; Fusarium species; Aspergillus species; Penicillium species; or any combination thereof.
In some embodiments, the present description relates to metabolites from: Paenibacillus polymyxa; Paenibacillus peoriae; Bacillus amyloliquefaciens; Burkholderia cepacia; Mortierella alpine; Giberella moniliformis; Fusarium oxysporum; Aspergillus niger Tiegh; Aspergillus hiratsukae; Penicillium ochrochloron; or any combination thereof. In a more particular embodiment, the metabolites may be from Burkholderia cepacia, Paenibacillus polymyxa; Paenibacillus peoriae; and/or Bacillus amyloliquefaciens. In a more particular embodiments, the metabolites may be from: Paenibacillus polymyxa T1 B; Paenibacillus polymyxa 44; Paenibacillus sp. 62; Paenibacillus polymyxa 273 (since renamed as Paenibacillus peoriae 273); Paenibacillus polymyxa 329; Paenibacillus sp. 344; Paenibacillus polymyxa 390; Paenibacillus polymyxa To99 (NRRL B-67020; since renamed as Paenibacillus peoriae To99); Paenibacillus polymyxa TP12; Paenibacillus polymyxa TP29; Paenibacillus polymyxa TP77; Paenibacillus polymyxa V25T; Paenibacillus polymyxa TFr60; Paenibacillus sp. TFr101 (NRRL B- 67019; since renamed as Paenibacillus peoriae TFr101); Paenibacillus polymyxa TAu1 ; Paenibacillus polymyxa TM54; Bacillus amyloliquefaciens subsp. plantarum 16; Bacillus amyloliquefaciens subsp. plantarum 33; Bacillus amyloliquefaciens subsp. plantarum 71 (NRRL B-67021); Bacillus amyloliquefaciens subsp. plantarum 237; Bacillus amyloliquefaciens subsp. plantarum 335; Bacillus amyloliquefaciens subsp. plantarum VFb49; Burkholderia cepacia BC19; Burkholderia cepacia BC153; Mortierella sp. VFb1 ; Giberella sp. TFr4; Fusarium sp. FI3S; Aspergillus sp. 8PT; Aspergillus sp. FG; Penicillium sp. VFr37; or any combination thereof.
In some embodiments, the present description relates to metabolites that may have further antimicrobial, bactericidal, and/or fungicidal activity against a plant and/or a human pathogenic microorganism, such as a virus, bacteria, fungus, yeast, mold, or any combination thereof. In some embodiments, the antimicrobial activity may comprise antagonistic activity. As used herein the terms "bactericidal" or "fungicidal" refers to the ability of a composition or substance to increase mortality or inhibit the growth rate of bacteria or fungi, respectively. In some embodiments, the pathogenic microorganism may be: Xanthomonas euvesicatoria; Xanthomonas fraganae; Xanthomonas perforans; Xanthomonas campestns; Xanthomonas gardnen, Pseudomonas syringae; Erwinia amylovora; Burkholderia glumae; Escherichia coli; Bacillus subtilis; Staphylococcus aureus; Pseudomonas aeruginosa; or any combination thereof. In a more particular embodiment, the pathogenic microorganism may be: Xanthomonas euvesicatoria R4; Xanthomonas gardneri DC00T7A; Xanthomonas fraganae LMG 708; Pseudomonas syringae DC3000; Erwinia amylovora 435; Burkholderia glumae LMG10905; Escherichia coli 0157:H7 EDL933; Bacillus subtilis ED66; Staphylococcus aureus ED711; Pseudomonas aeruginosa PA416A; or any combination thereof. In some embodiments, "mold" refers to a fungus that grows in the form of multicellular filaments called hyphae.
In some embodiments, the present description relates to metabolites that may exhibit higher antimicrobial activity against Xanthomonas species, as compared to other species (e.g., non-Xanthomonas species). In some embodiments, the metabolites of the present description are specifically active against Xanthomonas species. In used herein, "specifically active" means that metabolites of the present description show relatively higher antimicrobial activity against Xanthomonas species, than phytopathogenic non-Xanthomonas species.
In some embodiments, the present description relates to metabolites that may comprise lipopeptides and/or siderophores having anW-Xanthomonas activity such as lipopeptides and/or siderophores from Bacillus and/or Paenibacillus. In some embodiments, the lipopeptides may be non-ribosomal lipopeptides (NRPs). In some embodiments, the lipopeptides and/or siderophores may comprise one or more of surfactin, fengycin, plipastatin, iturin, bacilysin, bacillibactin, bacillomycin, locillomycin, paenilarvin, pelgipeptin, polymyxin, paenibacterin, fusaricidin, bacitracin, and tridecaptin. In some embodiments, the metabolites may comprise plipastatin and/or locillomycin. In some embodiments, the compositions of the present description may further comprise an agriculturally acceptable excipient. As used herein, the phrase "agriculturally acceptable excipien refers to an essentially inert substance that can be used as a diluent and/or carrier for an active agent (e.g., antimicrobial metabolites of the present description) in a composition for treatment of plants. In some embodiments, the compositions of the present description may further comprise one or more of: non-toxic carriers, surfactants, preservatives, nutrients, UV protectants, stickers, spreaders and chelating agents.
In some embodiments, the compositions of the present description may lack viable cells of the bacterial and/or fungal species from which the antimicrobial metabolites originate. In some embodiments, the compositions of the present description may be a cell-free composition (e.g., a cell-free supernatant). Cells can be removed by, for example, filtration and/or centrifugation. In some embodiments, the compositions of the present description may comprise killed cells from the bacterial and/or fungal species. In some embodiments, the compositions of the present description may comprise viable cells and/or spores from the bacterial and/or fungal species.
In some embodiments, the compositions of the present description may be in the form of a liquid, concentrate, powder, tablet, gel, paste, pellets, granules, or any combination thereof.
In some embodiments, the compositions of the present description, once applied to a target plant, may have no detectable phytotoxic effect on the target plant, or on the fruits, nuts, or leaves thereof, as compared to a control plant that is untreated (e.g., treated with water or with another agent).
In some embodiments, the compositions comprise an effective amount of antimicrobial metabolites of the present description. An "effective amount', as used herein, is an amount sufficient to effect beneficial or desired results. An effective amount can be administered in one or more administrations. In terms of treatment, inhibition or protection, an effective amount is that amount sufficient to ameliorate, stabilize, reverse, slow or delay progression of the target infection or disease states.
In some embodiments, the compositions may comprise at least 100 ppm, 200 ppm, 300 ppm, 400 ppm, 500 ppm, 600 ppm, 700 ppm, 800 ppm, 900 ppm, 1000 ppm, 1500 ppm, 2000 ppm, 2500 ppm, 3000 ppm, 3500 ppm, 4000 ppm, 4500 ppm, 5000 ppm, 5500 ppm, 6000 ppm, 6500 ppm, 7000 ppm, 8000 ppm, 8500 ppm, 9000 ppm, or 9500 ppm of the metabolites of the present description. In some embodiments, the compositions may comprise between about 100 ppm, 200 ppm, 300 ppm, 400 ppm, 500 ppm, 600 ppm, 700 ppm, 800 ppm, 900 ppm, 1000 ppm, 1500 ppm, 2000 ppm, 2500 ppm, 3000 ppm, 3500 ppm, 4000 ppm, 4500 ppm, 5000 ppm, to about 10 000 ppm of said metabolites of the present description. The term "about' is used herein to indicate that a value includes the standard deviation of error for the device or method being employed to determine the value. In general, the terminology "about" is meant to designate a possible variation of up to 10%. Therefore, a variation of 1 , 2, 3, 4, 5, 6, 7, 8, 9 and 10% of a value is included in the term "about".
In some embodiments, compositions of the present description may be used as an anti-microbial (e.g., bactericidal and/or fungicidal) agent against a plant and/or human pathogenic microorganism, or for the manufacture of an anti-microbial agent for same. In some embodiments, the pathogenic microorganism may be a Xanthomonas species. In some embodiments, antimicrobial compositions, bacterial or fungal strains of the present description may be used for biological control. As used herein, the expression "biological control" refers to the control of a pathogen or any other undesirable organism by the use of at least a second organism other than man. An example of known mechanisms of biological control is the use of microorganisms that control root rot by out-competing fungi for space on the surface of the root, or microorganisms that either inhibit the growth of or kill the pathogen. The "host plant' in the context of biological control is the plant that is susceptible to disease caused by the pathogen. In the context of isolation of an organism, such as a bacterium or fungal species, from its natural environment, the host plant is a plant that supports the growth of the bacterium or fungus, for example, a plant of a species the bacterium or fungus is an endophyte of.
In some embodiments, the compositions defined herein may be a biopesticide or biological pesticide. As used herein, the expressions "biopesticide" and "biological pesticide" refer to non-naturally occurring commercial products that include naturally occurring metabolites/microorganisms which are formulated to have anti-microbial activity when applied to plants. Such formulations may increase the stability and/or concentrations of the metabolites/miroorganisms, which enable them to be useful as plant pesticides.
In some embodiments, the present description relates to an isolated strain of antimicrobial bacterial or fungal strains as defined herein. As used herein, an "isolated" strain of a microbe is a strain that has been removed from its natural milieu. As such, the term "isolated" does not necessarily reflect the extent to which the microbe has been purified. But in different embodiments an "isolated" culture has been purified at least 2x, 5x, 10x, 50x or 100x from the raw material from which it is isolated. As a non-limiting example, if a culture is isolated from soil as raw material, the organism can be isolated to an extent that its concentration in a given quantity of purified or partially purified material (e.g., soil) is at least 2x, 5x, 10x, 50x or 100x that in the original raw material. A "substantially pure culture" of the strain of microbe refers to a culture which contains substantially no other microbes than the desired strain or strains of microbe. In other words, a substantially pure culture of a strain of microbe is substantially free of other contaminants, which can include microbial contaminants as well as undesirable chemical contaminants. Further, as used herein, the expression "enriched culture" of an isolated microbial strain refers to a microbial culture wherein the total microbial population of the culture contains more than 50%, 60%, 70%, 80%, 90%, or 95% of the isolated strain.
In some embodiments, the present description relates to genetically modified or mutant antimicrobial bacterial or fungal strains as defined herein. As used herein, the term "mutant" making reference to a microorganism refers to a modification of the parental strain in which the desired biological activity (e.g., ability to produce antimicrobial metabolites as defined herein) is similar to or higher than that expressed by the parental strain.
In some embodiments, compositions of the present description may be applied to a growing plant, such as a fruit plant, nut, cereal, vegetable, or flower. Examples of fruit plants include: apple, apricot, banana, blackberry, blueberry, cantaloupe, cherry, cranberry, currant, grapes, greengage, gooseberry, honeydew, lemon, mandarin, melon, orange, peach, pears, pineapple, plum, raspberry, strawberry, tomatoes, watermelon, grapefruit, pepper, olive, or lime. Examples of nuts include: almond, beech nut, Brazil nut, butternut, cashew, chestnut, chinquapin, filbert, hickory nut, macadamia nut, pecan, walnut, or pistachio. Examples of cereals include: amaranth, breadnut, barley, buckwheat, canola, corn, fonio, kamut, millet, oats, quinoa, cattail, chia, flax, kahiwa, pitseed goosefoot, wattleseed, rice, rye, sorghum, spelt, teff, triticale, wheat, or colza. Examples of vegetables include: artichoke, bean, beetroot, broad bean, broccoli, cabbage, carrot, cauliflower, celery, chicory, chives, cress, cucumber, kale, dill, eggplant, kohlrabi, lettuce, onion, pepper, parsnip, parsley, pea, potato, pumpkin, radish, shallot, soybean, spinach, turnip, or peanut. Examples of flowers include: species of the Euphorbiaceae (e.g., Euphorbia pulcherrima (poinsettia), Euphorbia milii (crown-of-thorns), Codiaeum variegatum (croton)), members of the family Rosaceae (RosoideaelRosa), Begoniaceae (Begonia), Araceae (e.g., Dieffenbachia, Anthurium, Philodendreae (Philodendron), and Caladieae (Syngonium), English ivy and other Araliaceae species, Pelargonium (geranium), Ficus, Hydrangea, Zinnia, ornamental Prunus species, ornamental Peppers, as well as other flowers and/or ornamental plants susceptible to infection by Xanthamonas.
In particular embodiments, compositions of the present description may be applied to a tomato plant, a pepper plant, a berry plant (e.g., strawberry plant), lettuce, a citrus plant, a walnut plant, a rice plant, a kiwi plant, or any combination thereof.
In particular embodiments, compositions of the present description may be applied to a plant cell or tissue or organ obtained from: a leaf, a stem, a flower, a fruit, a tuber, a rhizome, a corm, a root, or any combination thereof, or a part thereof.
In some aspects, the present description relates to a method for producing a composition as defined herein, the method comprising culturing viable cells from a suitable bacterial and/or fungal species to produce the metabolites; and harvesting the metabolites of the present description produced therefrom. The term "culturing", as used herein, refers to the propagation of organisms on or in media of various kinds.
The culture media and/or culture/fermentation conditions may be modified and/or optimized to increase the anti-Xanthomonas activity of the metabolites that are produced. In some embodiments, Landy medium or variations thereof may be used to enhance the anti-Xanthomonas activity of metabolites produced by B. amyloliquefaciens (e.g., subsp. plantarum isolates 71 and VFb49). Such medium may typically contain: glucose (e.g., 20 g/L), L- glutamic acid (e.g., 5.0 g/L), yeast extract (e.g., 1.0 g/L), K2HP04 (e.g., 1.0 g/L), MgS04 7H20 (e.g., 0.5 g/L), KCI (e.g., 0.5 g/L), CuS04 (e.g., 1.6 mg/L), Fe2(S04)3 (e.g., 1.2 mg/L), MnS04 (e.g., 0.4 mg/L). Without being bound by theory, Landy medium or variations thereof may facilitate the production of polyketides and lipopeptides (Chen et al., 2009), as well as to enhance the production of biosurfactant (e.g., Ben Ayed, Jemil et al. 2015). Furthermore, without being bound by theory, Landy medium or variations thereof may increase the production of fusaricidin-family antibiotics (e.g., Vater et al., 2015) and/or siderophore bacillibactin (e.g., Li et al., 2014). In some embodiments, tryptic soy broth (TSB) (Kim et al., 2015) or variations thereof may be used to enhance the
Figure imgf000015_0001
activity of metabolites produced by P. polymyxa isolates (e.g., To99, TFr101 and 273). In some embodiments, fermentation may be conducted at about 30°C (e.g., with shaking at 250 rpm). Without being bound by theory, optimization of the culture media and/or culture/fermentation conditions may improve the production and/or relative concentrations of lipopeptides (e.g., surfactin, fengycin, and/or iturin) and/or other metabolites having anW-Xanthomonas activity.
In some aspects, the present description relates to an antimicrobial composition produced by the aforementioned method.
In some aspects, the present description relates to a method for controlling the growth of a pathogenic microorganism on a target plant or tissue, the method comprising contacting the target plant or tissue with the composition of the present description. In some embodiments, the contacting may comprise spraying, irrigating, painting, daubing, and/or fogging, onto and/or into the target plant or tissue, the target plant or tissue's hydroponic substrate, and/or the target plant or tissue's agricultural earth.
In some aspects, the present description relates to a kit for preparing an aqueous solution for use in controlling pathogens on a plant tissue and/or plant cells of a growing plant, the kit comprising the composition as defined herein, and a suitable container. In some embodiments, the container may be a pouch, a tablet, or a bucket. In some embodiments, the kit may be used for controlling infection by the pathogenic microorganism (e.g., Xanthomonas species) on a plant.
In some embodiments, the compositions of the present description may be used or formulated with other antimicrobial agents, such as one or more of the agents described in Table 1.
Table 1 : Commercially available biopesticides composed of active microorganisms
Figure imgf000015_0002
Figure imgf000016_0001
Company -
Mode of Registered and
Product: Bioagent Target pathogens Crop
action commercialized
-Reference
EDEN Bioscience
Messenger™: Field ornamental Corporation,
Plant
Erwinia amylovora Many and vegetable USA
activator
(HrpNharpin protein) crops McSpadden Gardner
B.B. 2002
Pseudomonas syringae
pv. syringae,
NacillusPro™: P. syringae pv. tomato,
Brevibacillus Xanthomonas campestris Tomato,
parabrevis strain N4, pv. vesicatoria, peppers, Bio InsumosNativa Bacillus subtilis strain Antibiosis, X. campestris pv. cucurbits, Ltda, Chili N5, Bacillus cereus competition coralina, walnut, peanut, Valdes et al., 2012 strain N6, Bacillus Xanthomonas juglandis, hop, leafy
cereus strain N7 Clavibacter michiganensis vegetables
sub sp. michiganensis,
Acetobacter sp.,
Erwinia caratovora
Bio-care Technology,
Australia/
Nogall™: Fruit, nut, and
Agrobacterium New BioProducts Inc., Agrobacterium Antibiosis ornamental
tumefaciens Australia, USA radiobacter K1026 nursery stock
McSpadden Gardner B.B. 2002
Grape, apples, AgraQuest Inc., pear, banana, Chile, USA, New
Xanthomonas spp. cherry, walnut, Zealand, Mexico, (bacterial spot), peanut, hop, Japan, Israel, Costa
Serenade®:
Xanthomonas leafy vegetables, Rica, Philippines, Bacillus subtilis
Antibiosis campestris tomato, peppers, Guatemala, Honduras, strain QST 713
(walnut blight), cucurbits, Argentina, Italy, Erwinia amylovora mango, bean, France, Turkey, (fire blight) onion, garlic, Switzerland, Korea, potato, broccoli, Ecuador, Peru carrot Cawoy H. et al., 2011
Although the present invention has been described hereinabove by way of specific embodiments thereof, it can be modified, without departing from the spirit and nature of the subject invention as defined in the appended claims. EXAMPLES
Example 1 :
Isolation and antagonistic/antimicrobial screening of microorganisms
1.1 Samples for isolation of microorganisms
Different plant organs (leaves, stems and fruits) and seeds of tomatoes, peppers, onions, corns, eggplants, strawberries and raspberries, as well as soil samples from agricultural fields were collected in 2011-2013 from various locations (Table 2).
Table 2: Characteristics of environmental samples collected for isolation of microorganisms
Figure imgf000018_0001
1.2 Isolation of microorganisms from environmental samples
Media. Three solid nonselective media (R2A, Tryptic Soy Agar (TSA) agar and Plate Count Agar) and three selective media (Benedict, BCSA and Gould) with cycloheximide (50 mg/mL) were used for isolation of bacteria from environmental samples. Potato Dextrose Agar (PDA) and Malt Extract Agar (MEA) with a mix of antibiotics (streptomycin, penicillin and chloramphenicol in concentrations of 1.0, 0.5 and 0.05 mg/mL, respectively) were used for isolation of microscopic fungi. Isolation of microorganisms from plants. Ten seeds and 3 segments (0.5 cm2) randomly cut out from each leaf, stem, root and fruit were resuspended by vortexing in 5 mL of 0.85% NaCI. Aliquots (100 μΙ_) of each suspension were spread on nonselective media and selective media plates. Plates for isolation of bacteria were incubated in the dark for 2 days at room temperature (approximately 21 °C). PDA and MEA plates were incubated in the dark for 7-10 days at 25±2°C [Roberts and Koenraadt, 2014; International Seed Federation, 2011 ; Remeeus and Sheppard, 2006; Yin et al., 2011 ; Pusey et al., 2009; Yoshida et al., 2001].
Isolation of microorganisms from soil and water. Suspensions were prepared with 1 g of soil or 1 mL of water added to 9 mL of Phosphate-Buffered Saline (PBS), under agitation for 30 min. For isolation of sporulating bacteria, soil suspensions were preheated at +80°C for 30 min. Sample suspensions were submitted to serial dilutions. Hundred μί of each dilution (102, 103 and 104) were spread on nonselective media and selective media plates for isolation of bacteria and fungi. Plates were incubated under the same conditions described above [Zanatta et al., 2007]. Isolation of microorganisms was performed in triplicate.
Preservation of isolated bacteria and fungi. Colonies of bacteria with different morphological characteristics were transferred to tubes containing 3 mL of Tryptic Soy Broth (TSB) and were cultivated overnight at 30°C. Bacteria were preserved in 15% of glycerol at -80°C for further manipulations.
A fragment of mycelium from a fungus colony which was morphologically different from others was picked up and transferred into a 20-mL tube with PDA slant, incubated at 25±2°C for 14 days to form a new colony, and stored at +4°C under a layer of mineral oil for further manipulations [Humber, 1997]. 1.3 Strains used to evaluate the antimicrobial activity of isolates
Pathogenic strains used as indicators for evaluating antagonistic and antimicrobial activity of bacterial and fungal isolates are the following: Xanthomonas campestris ED1985 was isolated from Sherrington lettuce leaves. Xanthomonas campestris 901 was received from MAPAQ-Agri-Reseau-Phytoprotection. Xanthomonas perforans T4 and Xanthomonas euvesicatoria R4 were provided by University of Florida, Plant Pathology Faculty (USA). Xanthomonas gardneri DC00T7A was provided by Agriculture and Agri-Food Canada (London, ON, Canada). Xanthomonas fragariae LMG 708 and Burkholderia glumae LMG10905 were received from the Belgian coordinated collections of microorganisms (BCCM). Erwinia amylovora 435 was provided by Institut de recherche et de developpement en agroenvironnement (IRDA, QC, Canada). Multi-drug resistant, clinical strains Pseudomonas aeruginosa PA416A and Staphylococcus aureus ED711, as well as Pseudomonas syringae DC3000 and Bacillus subtilis ED66 were used from the Deziel lab collection, INRS-lnstitut Armand-Frappier (Laval, QC, Canada). Escherichia coli 0157:H7 EDL933 was also tested. All strains are stored in 15% glycerol at -80°C. 1.4 First step of screening: Antagonistic activity assays
Antagonistic activity of bacterial isolates against Xanthomonas was determined by various Petri dish plate assays. Bacteria
Method 1 , Individual bacterial colonies were picked for each bacterial strain and incubated in 3 ml_ of Tryptic Soy Broth (TSB) overnight at 30°C. Five μΙ_ of each bacterial suspension was dropped on a lawn of target bacteria X. campestris ED1985 or X campestris ED740 (OD620 = 0.2) on TSA plates. Petri dishes were incubated at room temperature (approx. 21 °C) for 2 days. Bacterial strains which formed a clear halo (inhibition zone) around of the colonies on the lawn of X. campestris, (e.g. see Figure 2A and B) were selected for determination of antimicrobial activity against X. perforans T4, the second step of screening (see below).
Method 2. Bacterial colonies appeared on Petri dishes with nonselective and selective media after 2 days of incubation at room temperature (approx. 21 °C) were covered by a layer of 5 ml_ of Top Agar containing 100 μΙ_ of X. campestris ED1985 (OD620 = 0.2). Bacterial strains which formed a halo around of theirs colonies, zones of growth inhibition of X. campestris, (e.g., Figure 3) were selected for determination of antimicrobial activity against X. perforans T4, the second step of screening (see below).
Fungi
To estimate antagonistic activity of fungi against X. campestris, block-agar diffusion assay was used [Yoshida et al., 2001 ; Agarry, 2005]. First of all, 50 μΙ_ of X. campestris ED1985 (OD620 = 0.2) grown overnight in
TSB at 30°C and resuspended in sterile water was spread on TSA plates to obtain a lawn of pathogenic bacteria.
The mycelial plug (10 x 10 mm) of fungal colony appeared after 10 days of incubation at 25±2°C on PDA plate and was placed at the center of a lawn of X. campestris ED1985. All plates were incubated at room temperature (approx.
21 °C) for 2 days. After this period, the plates were checked for the presence or absence of X. campestris ED1985 growing inhibition halo, indicating the occurrence of antibiosis between the microorganisms tested (Figure 4). Fungal strains formed a halo were used for determination of antimicrobial activity against X. perforans T4, the second step of screening.
1.5 Second step of screening: Antimicrobial activity assays
For evaluation of extracellular antimicrobial activity, bacterial strains were cultivated in 3 ml_ of TSB at 30°C,
150 rpm for 2 days. Fungal strains were grown in 250 ml_ Erlenmeyer flasks containing 50 ml_ of Czapek-Dox Broth at 25±2°C, 150 rpm. After 10 days of incubation, fungal biomass was discarded. The cultures of bacteria and fungi were centrifuged at 18 000 x g for 10 min at 20°C. To get cell-free fermentation filtrate, the supernatants of bacterial and fungal strains were separately collected and filtered (0.22 μιτι pore diameter) [Velmurugan, 2009; Rashid and Khan, 2000].
The antimicrobial activity against X. perforans T4 was assessed using well-diffusion inhibition assay [Obradovic et al., 2002; WO/2012/051699; Lindow et al., 2003]. First, a lawn of indicator bacteria was produced on the surface of the agar plates. Fifty μΙ_ of X. perforans T4 (OD620 = 0.2) grown overnight in TSB at 30°C and resuspended in sterile water was spread on TSA plates. Wells were bored into the agar layer with a sterile glass tube (10 mm diameter) and filled with 200 μΙ_ of cell-free fermentation filtrate, taking care to avoid spillage on the surface of the medium. The plates were then incubated at room temperature (approx. 21 °C) and the inhibition diameter of X. perforans T4 growth around the wells was measured after 2 days. To establish the controls, 200 μΙ_ of TSB and Czapek-Dox liquid medium were added to the wells on the lawn of X. perforans T4 instead of cell-free fermentation filtrates. Plates were incubated under the same conditions described above. Three replicates were performed for each treatment.
1.6 Results
A total of 123 environmental samples (seeds, different vegetable tissues, and soil) from different geographic locations were used for isolation of microorganisms during the period from November 2011 to August 2013. About 5000 isolated bacterial and 333 fungal strains were tested against X. campestris ED740 and X. campestris ED1985 using antagonistic activity assay (the 1st step of screening), as described in Example 1.4.
According to the results of the 1st step of screening, 612 bacterial and 124 fungal strains showed inhibition zones with different areas for different strains and were considered as possessing antagonistic activity against X. campestris ED740 and X. campestris ED1985 (e.g., Figure 2A, 2B, Figure 3, and Figure 4). All these isolates were stored in glycerol at -80°C. These strains were chosen for estimation of their cell-free filtrate activity against both phytopathogenic indicators.
During the 2nd step of screening (Example 1.5), antimicrobial activity was observed in 108 bacterial and 6 fungal strains which showed growth inhibition zones with different areas for different strains (Table 2 and 3). Among them, cell-free filtrate of Paenibacillus and Bacillus species showed the strongest antimicrobial activity against X. perforans T4 and X. campestris ED740 no matter where these strains were isolated from.
Clear inhibition halos with diameter 29.0-33.0 mm were formed by cell-free filtrate of 3 bacterial and 3 fungal strains, and were retained for further analysis (Figure 5 and Figure 6 A, B). The same antimicrobial activity was shown against X. campestris ED740 (Table 3). The active strains described in Table 3 and 4 were identified using various techniques as will be discussed later. Table 3: Characteristics of the most active bacterial strains against Xanthomonas perforans T4
Figure imgf000022_0001
Figure imgf000023_0001
Nonselective media: R2A, Tryptic Soy Agar (TSA), Plate Count Agar (PCA). Selective mec ia: Burkholderia cepacia selective agar (BCSA), Benedict (for Steptomyces species), ± Standard Error of Mean (SEM), * Diameter of well is 10 mm.
Table 4: Characteristics of the most active fungal strains against Xanthomonas perforans
In summary, thousands of microorganisms were screened from 123 different environmental samples collected from different geographical areas for their activity against phytopathogenic Xanthomonas species. High anW-Xanthomonas activity was observed in 108 bacterial and 6 fungal isolates, which showed growth inhibition zones with different areas for different strains. The most active isolates were identified using microscopic diagnostic, biochemistry assays, fatty acid analysis and genes sequencing. Unexpectedly, most of the bacterial isolates having the highest activity against Xanthomonas species belonged to the genus Paenibacillus (16 isolates) and Bacillus (6 isolates). Interestingly, fifteen isolates of Paenibacillus were identified as Paenibacillus polymyxa and one isolate was identified as Paenibacillus jamilae. All six isolates of the genus Bacillus were identified as B. amyloliquefaciens subsp. plantarum. Thus, not only Paenibacillus species but also Bacillus species were found to be active against pathogenic Xanthomonas bacteria.
It is worth mentioning, that fungi and other bacteria such as Burkholderia (Tables 3 and 4) were also found to be active against Xanthomonas species. Thus, activity against Xanthomonas pathogenic strains is not limited to Bacillus and Paenibacillus species but it is most prevalent in these two species.
Example 2:
Identification of bacterial and fungal isolates
2.1 DNA extraction of the isolates
A 1.5 mL of overnight culture in TSB was pelleted in a microcentrifuge tube at 13000 rpm for 10 minutes. The supernatant was then discarded and the pellet was resuspended in 1 mL of extraction buffer (50 mM Tris-HCI, 5 mM EDTA, 3% SDS, pH 8). The resuspended cells were transferred in a microtube containing 150-200 mg of sterile glass beads. The tubes were then placed in the Fastprep™ for 50 seconds at a 4 m/s speed, for two rounds and put on ice for 2 minutes between the rounds. The broken cells were then centrifuged at 13000 rpm for 10 minutes and the supernatant was mixed with 1 :5 volume of ammonium acetate 10 N. The mixture was vortexed, placed on ice for 5 minutes, and centrifuged at 13000 rpm for 15 minutes at 4°C. The supernatant was placed on ice for 5 minutes and centrifuged again at 13000 rpm for 15 minutes at 4°C. The supernatant was then mixed with 1 : 1 volume of ice-cold isopropanol, and the DNA was precipitated for 1-2 hours at 4°C. The tubes were centrifuged at 13000 rpm for 15 minutes at 4°C and the supernatant was discarded. 500 μί of ice-cold 70% was added to the pellet to wash the precipitated DNA and centrifuged at 13000 rpm for 15 minutes at 4°C. The supernatant was discarded and the pellet was dried under the biosafety cabinet. The dry DNA pellet was resuspended in 50 μί of sterile ddhbO and kept at - 20°C. All the DNA samples were dosed and diluted to a final concentration of 50 ng/ L [Nakamura, 1987; Aguilera et al, 2001 ; Priest et al., 1987].
2.2 16S rRNA and 18S rRNA gene sequence analysis
In order to identify the isolates that showed the strongest anW-Xanthomonas perforans activity, PCR amplification of the gene coding for 16S rRNA (for 18 bacterial isolates) or 18S rRNA (for 6 fungal isolates) was performed using primers well described in the literature (Table 5) [Priest et al., 1987; Frank et al., 2008]. Table 5: Primers used to determine 16S and 18S rRNA gene sequences of bacterial and fungal isolates
Primers Target Sequence5'→3' Product size Reference
pA-27f-YM 16S rDNA AGAGTTTGATYMTGGCTCAG 1.6 kb Frank et al., 2008 pH AAGGAGGTGATCCARCCGCA
ITS1-F 18S rDNA, CTTG GTC ATTTAG AG G AAGTAA 750 bp Martin and Rygiewicz, 2005
ITS4 ITS regions TCCTCCGCTTATTGATATGC
PCR-amplifications were carried out in a 50-μΙ_ reaction mixture (Table 6) containing Feldan Taq DNA Polymerase (Bio Basic Canada Inc., Markham, Ontario, Canada).
Table 6: Reaction mixture
Reaction mixture
In a final volume of 50 μί.
1X Taq buffer
200 μΜ dNTPs mix
0.4 μΜ Primer F
0.4 μΜ Primer R
1 unit Taq DNA polymerase
50 ng DNA extract
The amplifications were performed in a C1000 Touch™ Thermal Cycler (Bio-Rad Laboratory Inc., Canada) using specific PCR temperature protocol (Table 7).
Table 7: PCR Program for amplification of 16S and 18S rRNA fragments of bacterial and fungal isolates
Step Protocol
16S 18S
1 5 min 95°C 5 min 95°C initial denaturation
2 30 s 95°C 30 s 95°C denaturation
3 40 s 55°C 30s 59°C annealing
4 1.5 min 72°C 50s 72°C elongation
5 Repeat steps 2 to 4 Repeat steps 2 to 4 29 times
6 10 min 72°C 10 min 72°C final elongation
7 oo 40c 00 40c stop PCR reaction and refrigerate DNA products
end end
After DNA amplification, PCR products were analyzed by agarose gel electrophoresis (1.0% of agarose, 100V, 60 min), DNA was stained by ethidium bromide (0.5 Mg/mL), and visualized under UV illumination.
All PCR products were purified on a 1 % agarose gel using a Gel extraction kit (Bio Basic Canada Inc., Markham, Ontario, Canada) and sent to the sequencing platform to Institut de recherches cliniques de Montreal (IRCM). The same primers were used for the initial PCR reaction and the sequencing reactions.
The obtained sequences of each isolate were processed with the BioEdit™ sequence alignment editor and analyzed using the basic local alignment search tool (BLAST) sequence alignment system (http://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn&PAGE TYPE=BlastSearch&LINK LOC=blasthome), using the 16S ribosomal RNA sequences for Bacteria and Archaea and the nucleotide collection (nr/nt) for Fungi databases. This search tool was developed by the National Center for Biotechnology Information (NCBI) (US). If the sequence identity is above 99%, we could conclude that bacterial isolate belongs to the same species, and if the identity is higher than 97, strains are classified into the same genus or the same family.
A 1.6 kb 16S rRNA and 750 bp 18S rRNA and ITS regions fragments were amplified and sequenced from bacterial and fungal isolates respectively. Twenty-four bacterial and six fungal isolates were identified by gene sequencing (Table 8). Thus, using the BLAST alignment system, 2 bacterial isolates belong to the Burkholderia cepacia complex, 16 isolates belong to the Paenibacillus genus and 6 isolates belong to the Bacillus subtilis group. The 6 fungal isolates were identified as Mortierella alpina Peyronel; Gibberella moniliformis Wineland; Fusarium oxysporum Schltdl; Aspergillus niger Tiegh; Neosartorya hiratsukae Udagawa, Tsub. & Y. Horie (Aspergillus hiratsukae); and Penicillium ochrochloron Biourge (Table 8).
BLAST analysis of pairwise alignment of 16S rDNA sequences were highly similar to each other within the same genus and could not be used to distinguish different but closely related bacterial species, as P. polymyxa, P. peoriae, P. jamilae and P. kribbensis (Table 8). Thus, the next step was the strain identification by sequencing of specific genes of Burkholderia cepacia complex, Paenibacillus species and Bacillus subtilis group.
Table 8: 16S and 18S rRNA gene sequence identification of bacterial and fungal isolates
Figure imgf000026_0001
71 Bacillus subtilis group 98%
16 Bacillus subtilis group 99%
237 Bacillus subtilis group 99%
Fungi
VFb1 Mortierella alpina Peyronel 99%
TFr4 Gibberella moniliformis Wineland 99%
FI3S Fusarium oxysporum Schltdl. 99%
8PT Aspergillus n/ /er Tiegh. 99%
FG Neosartorya hiratsukae Udagawa, Tsub. & Y. Horie (Aspergillus hiratsukae) 99%
VFr37 Penicillium sp., Penicillium ochrochloron Biourge 99%-98%
2.3 Amplification of specific genes from Burkholderia and Paenibacillus species
BURKHOLDERIA
Isolates 19 and 153 of the Burkholderia cepacia complex were identified by recA sequence analysis. Fragment of recA gene was amplified in a 25-μΙ_ reaction mixture (Table 9) using recA_FS/recA_RS primer pairs (Table 10) and specific temperature protocol (Table 11). After DNA amplification, recA fragments were analyzed by agarose gel electrophoresis, stained by ethidium bromide, and visualized under UV illumination.
Gel electrophoresis of PCR products was done using 1.0% agarose in the presence of a molecular size standard (GeneRuler™ 1 Kb DNA Ladder, Fermentas) and sent for sequencing. Burkholderia cepacia ATCC 25416 was used as positive control. The same primers were used for the initial PCR reaction and the sequencing reactions.
The obtained sequences of recA gene were processed with the BioEdit™ sequence alignment editor and analyzed using NCBI BLAST sequence alignment system
(http://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome).
Table 9: Reaction mixture
In a final volume of 25 L
1X Taq buffer
200 μΜ dNTPs mix
0.4 μΜ Primer(s)F
0.4 μΜ Primer(s) R
1 unit Taq DNA polymerase
50 ng Bacterial DNA extract
Table 10: Primers used to determine the recA gene of Burkholderia cepacia complex isolates
Product
Primers Target Sequence 5'→3' Reference
size
recA Baldwin et al., recA_FS TGACCGCCGAGAAGAGCAA 390 bp
gene 2005 recA_RS GACCGAGTCGATGACGAT Table 11 : Temperature program for amplification of recA DNA fragments of Burkholderia isolates
Step Protocol
1 5 min 95°C initial denaturation
2 30 s 95°C denaturation
3 45 s 58°C annealing
4 1 min 72°C elongation
5 Repeat steps 2 to 4 29 times
6 10 min 72°C final elongation
7 oo 40c stop PCR reaction and refrigerate DNA products
8 end
Based on the 390 bp sequence of recA gene, bacterial isolates 19 and 153 showed 99% similarity with Burkholderia cepacia strains from GeneBank data base of NCBI. Thus, they were identified as B. cepacia (Figure 7).
PAENIBACILLUS
In order to refine the identification of Paenibacillus sp. isolates, the rpoB gene was amplified using the primer pair rpoB1698f / rpoB2041 r (Table 12). PCR-amplifications were carried out in a 25-μΙ_ reaction mixture (Table 13). The amplifications were performed using specific PCR temperature protocol (Table 14).
Table 12: Primers used to determine the rpoB gene of Paenibacillus isolates
Product
Primers Target Sequence 5'→3' Reference size
Dahllof et al., rpoB\ 698f rpoB gene AACATCGGTTTGATCAAC 240 bp 2000; da Mota et al., 2005 rpoS2041 r CGTTGCATGTTGGTACCCAT
Table 13: Reaction mixture
In a final volume of 25 μΙ_
1X Taq buffer
200 μΜ dNTPs mix
0.4 μΜ Primer rpoB\ 698f
0.4 μΜ Primer rpoS2041 r
1 unit Taq DNA polymerase
50 ng Bacterial DNA extract Table 14: Temperature program for amplification of rpoB DNA fragments of Paenibacillus isolates
Step Protocol
1 5 min 95°C initial denaturation
2 30 s 95°C denaturation
3 40 s 55°C annealing
4 35 s72°C elongation
5 Repeat steps 2 to 4 29 times
6 10 min 72°C final elongation
7 oo 40c stop PCR reaction and refrigerate DNA products
8 end
After DNA amplification, rpoB fragments were analyzed by agarose gel electrophoresis, stained by ethidium bromide and visualized under UV illumination.
All PCR products were purified on a 1 % agarose gel using a Gel extraction kit (Bio Basic Canada Inc.,
Markham, Ontario, Canada) and sent for sequencing. The same primers were used for the initial PCR reaction and the sequencing reactions. The obtained sequences of the rpoB gene of Paenibacillus sp. were processed with the BioEdit™ sequence alignment editor and analyzed using the basic local alignment search tool (BLAST) sequence alignment system:
(http://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome).
Based on the sequence of the rpoB gene, 16 isolates of Paenibacillus sp. were divided in three groups based on their similarity with species from GeneBank data base (NCBI). The first group includes four isolates 44, To99, TM54 and TP29 which showed 100% similarity with P. polymyxa. The second group represents nine isolates that had 99% nucleotide identity with P. polymyxa. Thus, thirteen isolates were preliminary identified as Paenibacillus polymyxa. The third group includes isolates 62, 344 and TFr101 showed 94-96% of similarity with P. polymyxa and P. peoriae (Table 15).
Since the identification of these 3 isolates based on rpoB DNA sequence identification were not clear enough, biochemical methods using Biolog™ microbial identification system (BIOLOG Inc., Hayward, USA) and fatty acid analysis (Keystone Labs Inc., Edmonton, Alberta, Canada) were performed to refine the identification.
Table 15. Identification of Paenibacillus sp. isolates by rpoB DNA sequencing
Isolate Microorganism % of similarity
62 Paenibacillus polymyxa/P. peoriae 95-96%
344 Paenibacillus polymyxa/P. peoriae 94-96%
TFM01 Paenibacillus polymyxa/P. peoriae 95-96%
V25T Paenibacillus polymyxa 99%
TP77 Paenibacillus polymyxa 99%
329 Paenibacillus polymyxa 99%
T1 B Paenibacillus polymyxa 99%
TP12 Paenibacillus polymyxa 99%
390 Paenibacillus polymyxa 99% 273 Paenibacillus polymyxa 99%
TAu1 Paenibacillus polymyxa 99%
44 Paenibacillus polymyxa 100%
To99 Paenibacillus polymyxa 100%
TFr60 Paenibacillus polymyxa 99%
TM54 Paenibacillus polymyxa 100%
TP29 Paenibacillus polymyxa 100%
2.3a Bioloq™ analyses of Paenibacillus species
To refine the identification of Paenibacillus species, the biochemical test system Biolog™ GENIII MicroPlate (OmniLog) was used. It allows analyzing a microorganism in 94 phenotypic tests: 71 carbon source utilization assays and 23 chemical sensitivity assays. Several isolates from each group mentioned above were chosen for performing this test. Well known Paenibacillus polymyxa and P. peonae strains (P. polymyxa ATCC7070, P. polymyxa CR1 , P. peoriae LMG 16104, P. peonae LMG 16111), as well as type strains: P. polymyxa LMG 13294, P. peoriae LMG 14832 and P. jamilae LMG 21667 were used for comparison with our isolates.
The isolates to be identified were grown on TSA and then resuspended in a special gelling inoculating fluid. Then, bacterial cell suspension was inoculated into the GENIII MicroPlate and incubated at 34 °C for 24 h to allow the phenotypic fingerprint to form. After incubation, the phenotypic fingerprints of purple wells were compared within isolates, known Paenibacillus polymyxa and P. peoriae strains and type strains of these species.
The carbohydrate utilization capabilities and chemical sensitivity of bacterial isolates are shown in Table 16. In general, obtained results of 94 biochemical tests revealed 11 differences between bacterial isolates and P. polymyxa LMG 13294 type strain, while there were less differences between the presently tested isolates and P. polymyxa CR1 (non type strain), as well as P. jamilae LMG 21667 (type strain). In two cases, utilization of methyl pyruvate, L-malic acid, bromo-succinic acid, and sensitivity to pH 5, 4% NaCI, rifamycin SV, lithium chloride varied within strains of tested bacterial isolates, type strains and well known non type strains (Table 16).
Table 16: Phenotypic characterization of Paenibacillus isolates and known Paenibacillus strains
Figure imgf000031_0001
Glycyl-L-Proline L-Alanine
L-Arginine
L-Aspartic Acid
L-Glutamic Acid
L-Histidine
L-Pyroglutamic Acid L-Serine
Pectin
D-Galacturonic Acid L-Galactonic Acid Lactone D-Gluconic Acid
D-Glucuronic Acid
Glucuronamide
Mucic Acid
Quinic Acid
D-Saccharic Acid p-Hydroxy-Phenylacetic Acid Methyl Pyruvate
D-Lactic Acid Methyl Ester L-Lactic Acid
Citric Acid
a-Keto-Glutaric Acid D-Malic Acid
L-Malic Acid
Bromo-Succinic Acid Tween 40
γ-Amino-Butryric Acid a-Hydroxy-Butyric Acid -Hydroxy-D,L-butyric Acid a-Keto-Butyric Acid Acetoacetic Acid
Propionic Acid
Acetic Acid
Formic Acid
chemical sensitivity assays pH 6
pH 5
1 % NaCI
4% NaCI
8% NaCI
1 % Sodium Lactate Fusidic Acid
D-Serine
Troleandomycin
Rifamycin SV
Minocycline
Li neomycin
Guanidine HCI
Niaproof 4 Vancomycin
Tetrazolium Violet
Tetrazolium Blue
Nalidixic Acid
Lithium Chloride + + - - - + + + - + - - + + +
Potassium Tellurite + + + + / + + + + + + + + + + +
Aztreonam + + - - -
Sodium Butyrate + + + + + + + + + + + + + + + +
Sodium Bromate . / . . . . . . . . .
+:positive result; -: negative result; /: borderline result (BIOLOG™, OmniLog)
In order to more easily understand these results, the percentage of similarity between isolated bacteria and the most closely related references strains P. polymyxa CR1 , P. jamilae LMG 21667 (type strain) and P. peoriae LMG 16111 (type strain) was calculated (Table 17). Only the highest percentage of similarity for each isolate is indicated in this table. Thus, isolates 62, 344, TFr101 and TP12 showed the highest similarity (about 97%) to P. jamilae LMG 21667. The isolates To99, 399 and V25T were more similar to P. polymyxa CR1 with 97.5% and 95.7% of similarity, respectively. Bacterial isolates 329 and 273 showed the same similarity (96.8%) to P. polymyxa CR1 and P. jamilae LMG 21667 (Table 17). Due to these differences, fatty acid analyses were performed for further bacterial isolate identification.
Table 17: Similarity of Paenibacillus sp. isolates with the most closely related reference strains based on their phenotypical characteristics
BIOLOG identification of Paenibacillus sp. isolates
Isolate Microorganism % of similarity
62 Paenibacillus jamilae 97.9%
Paenibacillus polymyxa 93.6%
344 Paenibacillus jamilae 96.8%
Paenibacillus polymyxa 94.7%
TFM01 Paenibacillus jamilae 98.9%
Paenibacillus polymyxa 94.7%
V25T Paenibacillus polymyxa 95.7%
Paenibacillus jamilae 93.6%
329 Paenibacillus polymyxa, 96.8%
Paenibacillus jamilae 96.8%
Paenibacillus peoriae 95.7%
390 Paenibacillus polymyxa 97.9%
Paenibacillus jamilae 93.6%
273 Paenibacillus polymyxa, 96.8%
Paenibacillus jamilae 96.8%
Paenibacillus peoriae 91.5%
To99 Paenibacillus polymyxa 97.9%
Paenibacillus jamilae 95.7%
TP29 Paenibacillus jamilae 96.8%
Paenibacillus polymyxa 94.7% The percentage of similarity is calculated as follows: (number of similar characteristics/total number of tests (94))Ί00.
2.4 Fatty acid analyses of Paenibacillus isolates
Fatty acids analysis was performed to confirm the identification of the Paenibacillus isolates (Keystone Labs Inc., Edmonton, Alberta, Canada). The fatty acids analysis is accomplished with the MIDI™ method (which analyzes fatty acid methyl esters in bacterial samples by gas chromatography) and then compared with the Sherlock libraries consisting in more than 100,000 analyses of strains obtained from experts and from culture collections (www.midi- inc.com). Microbial identification results from the MIDI system are expressed as a similarity index. The similarity index (SI) is a numerical value which is an expression of the relative distance from the population mean. An exact match of the fatty acid makeup of the unknown and the mean of the library entry would result in a SI of 1.000. As each fatty acid varies from the mean percentage, the SI will decrease in proportion to the cumulative variance between the composition of the unknown and the library entry.
Identification of Paenibacillus isolates based on the analysis of their fatty acid content was performed with the aim to distinguish closely related species within the same genus (Table 14). Based on the data of SI of fatty acid profiles, all bacterial isolates were identified as Paenibacillus polymyxa (SI ranged 0.7-0.9 ) with the exception of isolate TP29 with the lowest SI (0.598) that is closely related to Rothia dentocariosa (Table 18). According to the literature, this species is part of the normal community of microbes residing in the mouth and respiratory tract, while bacterial isolate TP29 was isolated from soil samples. Of note, there is no entry in the database to compare the isolates with the Paenibacillus jamilae strains. In fact, the fatty acid analysis of the P. jamilae LMG 21667 type strain gives an identification close to P. polymyxa (SI = 0.632) and to Arthrobacter globiformis (SI = 0.617).
Table 18: Identification of Paenibacillus isolates based on the analysis of their fatty acid content
Isolate Identification Similarity index
62 Paenibacillus polymyxa 0.808
Arthrobacter globiformis GC subgroup A 0.676
344 Paenibacillus polymyxa 0.799
Arthrobacter globiformis GC subgroup A 0.676
TFM01 Paenibacillus polymyxa 0.861
Arthrobacter globiformis GC subgroup A 0.675
329 Paenibacillus polymyxa 0.777
Artrhobacter globiformis GC subgroup A 0.622
Rothia dentocariosa 0.473
273 Paenibacillus polymyxa 0.934
Arthrobacter globiformis GC subgroup A 0.692
To99 Paenibacillus polymyxa 0.865
Arthrobacter globiformis GC subgroup A 0.685
V25T Paenibacillus polymyxa 0.815
Arthrobacter globiformis GC subgroup A 0.723 TP29 Rothia dentocariosa 0.598
Brevibacterium liquefaciens 0.593
Paenibacillus polymyxa 0.572
Arthrobacter globiformis GC subgroup A 0.381
2.5 Amplification of specific genes from Bacillus species
The rpoB, gyrA and gyrB gene fragments were used as molecular diagnostic markers to specifically identify bacterial isolates within the Bacillus subtilis group. To this end, specific primers for amplification of each gene were used (Table 19). PCR-amplifications were carried out in a 25-μΙ_ reaction mixture (Table 20). The amplifications were performed using specific PCR temperature protocol (Table 21).
After DNA amplification, rpoB, gyrA and gyrB fragments were analyzed by agarose gel electrophoresis, stained by ethidium bromide, and visualized under UV illumination.
All PCR products were purified on a 1 % agarose gel using a Gel extraction kit (Bio Basic Canada Inc., Markham, Ontario, Canada). The rpoB fragments were cloned into a pGEM-T-Easy vector™ (pGEM-t easy kit, Promega, Medison, USA) and sent for sequencing using the Sp6 and T7 primers (Table 19). The gyrA fragments were sequenced with the same primers that were used for the initial PCR reaction. The gyrB fragments were amplified with the universals UP-1 and UP-2r primers and sequenced with the UP-1 S and the UP-2Sr primers (Table 19).
The obtained sequences were processed with the BioEdit sequence alignment editor and analysed by NCBI
BLAST program:
http://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome).
Table 19: Primers used to determine specific genes of Bacillus isolates
Produ
Primers Target Sequence 5'→3' ct size Ref.
(bp)
rpoB-f AGGTCAACTAGTTCAGTATGGAC De Clerck et al., rpoB gene 579 2004; Gonzalez et rpoB-r AAGAACCGTAACCGGCAACTT
al., 2013 gyrA-f CAGTCAGGAAATGCGTACGTCCTT De Clerck et al., gyrA gene 1025 2004; Gonzalez et gyrA-r CAAGGTAATGCTCCAGGCATTGCT
al., 2013
GAAGTCATCATGACCGTTCTGCAYGCNGGNGG Wang et al., 2010;
UP-1
gyrB gene NAARTTYGA Yamamoto and
1200
(amplification) AGCAGGGTACGGATGTGCGAGCCRTCNACRTC Harayama, 1995 & UP-2r
NGCRTCNGTCAT 1998
UP-1 S gyrB gene GAAGTCATCATGACCGTTCTGCA Yamamoto and
1200
UP-2Sr (sequencing) AGCAGGGTACGGATGTGCGAGCC Harayama, 1995
Sp6 pGEM-T-Easy AGC TAT TTA GGT GAC ACT ATA G
Promega, 2010 T7 vector TTG TAA TAC GAC TCA CTA TAG GG Table 20: Reaction mixture
In a final volume of 25 μΙ_
1X Taq buffer
200 μΜ dNTPs mix
0.4 μΜ Primer F
0.4 μΜ Primer R
1 unit Taq DNA polymerase
50 ng Bacterial DNA extract
Table 21 : Temperature program for amplification of DNA fragments of Bacillus subtilis group specific genes by PCR
Step Protocol
rpoB gyrA gyrB
1 5 min 95°C 5 min 95°C 5 min 95°C initial denaturation
2 1 min 95°C 30 s 95°C 1 min 95°C denaturation
3 1 min 51°C 45 s 51 °C 1 min 60°C annealing
4 1 min 72°C 1 min 72°C 2 min 72°C elongation
5 Repeat steps 2 to 4 Repeat steps 2 to 4 Repeat steps 2 to 4 29 times
6 10 min 72°C 10 min 72°C 10 min 72°C final elongation
7 oo 40c 00 40c 00 40c stop PCR reaction and
refrigerate DNA products
8 end end end Based on gyrA sequence analysis, all six isolates of Bacillus subtilis group showed the closet similarity relative to Bacillus amyloliquefaciens, with sequence similarity of 99% and even 100% for isolates 71 and 237 (Table 22). Also, isolates 16 and 335 were high similar to B. velezensis that is a later heterotrophic synonym of B. amyloliquefaciens [Wang et al., 2008].
The partial sequence of the gene encoding the subunit B protein of DNA gyrase {gyrB) analysis and the sequence of rpoB DNA fragment showed the highest similarity (98-100%) of B. subtilis group isolates with B. amyloliquefaciens subsp. plantarum.
Thus, based on the results the sequencing of three specific genes from Bacillus subtilis group, all of the isolates belong to Bacillus amyloliquefaciens species and most likely to the plantarum subspecies.
Table 22: Identification of Bacillus sp. isolates based on sequencing of specific genes
Isolate % of similarity
gyrA gyrB rpoB
VFb49 B. amyloliquefaciens 99% - B. amyloliquefaciens subsp. plantarum 99%
33 B. amyloliquefaciens 99% B. amyloliquefaciens 98% B. amyloliquefaciens subsp. plantarum 99% subsp. plantarum
B. amyloliquefaciens 99% subsp. amyloliquefaciens
335 B. amyloliquefaciens 99% B. amyloliquefaciens 99% B. amyloliquefaciens subsp. plantarum 100% subsp. plantarum
B. velezensis 99%
16 B. amyloliquefaciens 99% B. amyloliquefaciens 99% B. amyloliquefaciens subsp. plantarum 100% subsp. plantarum
B. velezensis 99%
71 B. amyloliquefaciens 100% B. amyloliquefaciens 99% B. amyloliquefaciens subsp. plantarum 99% subsp. plantarum
B. amy/o/iquefaciens 99% subsp. amyloliquefaciens
237 B. amyloliquefaciens 100% B. amyloliquefaciens 100% B. amyloliquefaciens subsp. plantarum 100% subsp. plantarum
2.6 Detection of genes coding for metabolites by PCR
Different strains of B. amyloliquefaciens may produce enzymes and antibiotics, specially bacteriocins, lipopeptides and polyketides. In order to better characterize the B. amyloliquefaciens subsp. plantarum isolates, polymerase chain reactions were performed to determine the presence of metabolite genes involved in their biosynthesis. PCR-amplifications were carried out in a 25-μΙ_ reaction mixture (Table 23) with specific primers for each gene of interest (Table 24) and specific temperature protocol for each amplification reaction (Table 25). After DNA amplification, PCR products were analyzed by agarose gel electrophoresis (1.0% of agarose, 100V, 60 min), DNA was stained by ethidium bromide (0.5 pg/mL), and visualized under UV illumination.
Table 23: Reaction mixture
In a final volume of 25 μΙ_
1X Taq buffer
200 μΜ dNTPs mix
0.4 μΜ Primer F
0.4 μΜ Primer R
1 unit Taq DNA polymerase
Table 24: Amplification primers used to determine the presence of different metabolite genes in Bacillus sp. isolates
Primers Target Seguence5'→3' Product size Reference
OsboPI N Subtilosin CCTCATGACCAGGACTTCGCCTT 1220 bp Kabore et al., 2012
OsboP2N CGGTGCCGAGCGCTTCAGGT
SpaS_F Subtilin CAAAGTTCGATGATTTCGATTTGGATGT 152 bp Sutyak et al., 2008 SpaS R GCAGTTACAAGTTAGTGTTTGAAGGAA
Eric_F Ericin TCAACTGACCGGGCAGGAGC 1440 bp Kabore et al., 2012 Eric_R AAGTATTTGGCCTACAGCGACTCG
SUNT-F1 Sublancin GCTTTGTTAGAAGGGGAGGAAT 974 bp Chung et al., 2008 SUNT-R1 CTTGTCCCAACCCATAGGATAA
ITUC-F1 Iturin CCCCCTCGGTCAAGTGAATA 594 bp Chung et al., 2008 ITUC-R1 TTGGTTAAGCCCTGATGCTC
Athukorala et al., 2009;
ITUD1 F IturinA GATGCGATCTCCTTGGATGT 647 bp Ramarathnam et al.,
2007 ITUD1 R ATCGTCATGTGCTGCTTGAG
SRFA-F1 Surfactin AGAGCACATTGAGCGTTACAAA 626 bp Chung et al., 2008 SRFA-R1 CAGCATCTCGTTCAACTTTCAC
Am1-F Mycosubtilin CAKCARGTSAAAATYCGMGG 419 bp Tapi et al., 2010 Tm1-R CCDASATCAAARAADTTATC
Af2-F Fengycin GAATAYMTCGGMCGTMTKGA 542 bp Tapi et al., 2010 Tf1-R GCTTTWADKGAATSBCCGCC
Ap1-F Plipastatin AGMCAGCKSGCMASATCMCC 959 bp Tapi et al., 2010 Tp1-R GCKATWWTGAARRCCGGCGG
baeR_F Bacillaene ATGTCAGCTCAGTTTCCGCA 688 bp Compaore et al., 2013 baeR_R GATCGCCGTCTTCAATTGCC
mlnA_F Macrolactin CCGTGATCGGACTGGATGAG 668 bp Compaore et al., 2013 mnIA R CATCGCACCTGCCAAATACG
bacA/B_F Bacilysin TGCTCTGTTATAGCGCGGAG 910 bp Compaore et al., 2013 bacA/B_R GTCATCGTATCCCACCCGTC
bmyA_F Bacillomycin CTCATTGCTGCCGCTCAATC 853 bp Compaore et al., 2013 bmyA_R CCGAATCTACGAGGGGAACG
dfnA F Difficidin GGATTCAGGAGGGCATACCG 653 bp Compaore et al., 2013 dfnA R ATTGATTAAACGCGCCGAGC
Table 25: PCR program for amplification of DNA fragments of genes involved in
metabolite production in Bacillus sp. isolates
initial number of final Stop and
Step denaturation annealing elongation
denaturation cycles elongation refrigerate
Metabolite 1 2 3 4 5 6 7 8
Subtilosin 5 min 95°C 1 min 95°C 1 min 58°C 90 s 72°C 30 10 min 72°C ∞4°C end
Subtilin 5 min 95°C 1 min 95°C 1 min 55°C 30 s 72°C 30 10 min 72°C ∞4°C end
Ericin 5 min 95°C 1 min 95°C 1 min 58°C 90 s 72°C 30 10 min 72°C ∞4°C end
Sublancin 5 min 95°C 1 min 95°C 1 min 55°C 60 s 72°C 30 10 min 72°C ∞4°C end
Iturin 5 min 95°C 1 min 95°C 1 min 55°C 40 s 72°C 30 10 min 72°C ∞4°C end
IturinA 5 min 95°C 1 min 95°C 1 min 60°C 50 s 72°C 30 10 min 72°C ∞4°C end
Surfactin 5 min 95°C 1 min 95°C 1 min 55°C 50 s 72°C 30 10 min 72°C ∞4°C end
Mycosubtilin 5 min 95°C 1 min 95°C 1 min 45°C 30 s 72°C 30 10 min 72°C oo 40c end Fengycin 5 min 95°C 1 min 95°C 1 min 45°C 30 s 72°C 30 10 min 72°C 4°C end
Plipastatin 5 min 95°C 1 min 95°C 1 min 58°C 60 s 72°C 30 10 min 72°C 4°C end
Bacillaene 5 min 95°C 1 min 95°C 1 min 57°C 60 s 72°C 30 10 min 72°C 4°C end
Macrolactin 5 min 95°C 1 min 95°C 1 min 57°C 60 s 72°C 30 10 min 72°C 4°C end
Bacililysin 5 min 95°C 1 min 95°C 1 min 57°C 60 s 72°C 30 10 min 72°C 4°C end
Bacillomycin 5 min 95°C 1 min 95°C 1 min 57°C 60 s 72°C 30 10 min 72°C 4°C end
Difficidin 5 min 95°C 1 min 95°C 1 min 57°C 60 s 72°C 30 10 min 72°C 4°C end
As shown in Table 26, all the B. amyloliquefaciens subsp. plantarum isolates were found to be negative for the genes involved in the biosynthesis of subtilosin (ywiB, sboA), subtilin (spaS), ericin (eriC, eriSa, eriSb), sublancin (sun T), surfactin (srfA) and plipastatin (pps). Genes involved in the production of iturin (ituA) was detected in B. amyloliquefaciens subsp. plantarum isolates VFb49, 71 and 237. Only isolate 335 of this species does not have the gene involved in the biosynthesis of fengycin (fen). Gene involved in the production of bacilysin (bacAJB, bacB) was detected in B. amyloliquefaciens subsp. plantarum isolates VFb49, 237 and 16, while bacillomycin gene (bmyA) was detected in isolates VFb49, 237 and 16 of this species (Table 26). All isolates of B. amyloliquefaciens subsp. plantarum have genes involved in biosynthesis of iturinA (ituA), mycosubtilin (myc/itu), bacillaene (baeA), macrolactin (mnIA) and difficidin (dfnA) (Table 26). The detection of macrolactin, bacillomycin and difficidin genes is specific to B. amyloliquefaciens subsp. plantarum, since the primers were specifically designed for the type strain of this subspecies (B. amyloliquefaciens subsp. plantarum FZB42) [Compaore et al., 2013].
Table 26: Presence of metabolite production genes in Bacillus sp. isolates
Figure imgf000039_0001
Example 3:
Testing supernatants against different plant and human pathogens One strain of Bacillus and two strains of Paenibacillus species were selected for further analysis on crops (tomato and lettuce) because of their high antibacterial activity against pathogenic Xanthomonas bacteria. The supernatants of Bacillus amyloliquefaciens subsp. plantarum 71 , Paenibacillus sp. TFr101 , and Paenibacillus polymyxa To99 were tested against plant pathogens (X. perforans T4, X. campestris 901, X. euvesicatoria R4, X. gardneri DC00T7A, X. fragariae LMG 708, E. amylovora 435, B. glumae LMG10905, and P. syringae DC3000), as well as against B. subtilis ED66 and human pathogens (Escherichia coli 0157:H7 EDL933, and S. aureus ED711, P. aeruginosa PA416A), as shown in Table 27.
Table 27: Activity of bacterial supernatants against plant and human pathogens
Figure imgf000040_0001
Based on the results in Table 27, the supernatants of B. amyloliquefaciens subsp. plantarum 71 , P. polymyxa To99, and P. polymyxa TFr101 were highly active against all four Xanthomonas species tested. However, they were less active against other plant pathogenic bacteria such as B. glumae which causes panicle blight in rice, Erwinia amylovora which causes fire blight in apples and pears, and finally against Pseudomonas syringae that causes bacterial specks in many crops.
Some of the supernatants were also active against a species of Gram-positive Bacillus, as well as other human pathogens such as Escherichia coli 0157:H7 and Staphylococcus aureus. It is clear from above experiments that these supernatants produced by B. amyloliquefaciens subsp. plantarum 71 , P. polymyxa To99 and P. polymyxa TFr101 are specifically active against Xanthomonas species.
The cell free supernatants of B. amyloliquefaciens subsp. plantarum 71 , P. polymyxa To99 and P. polymyxa TFr101 stored for 1 year at -80°C were tested against plant and human pathogens. The zone of inhibition using the well diffusion test was equal to 25 mm in diameter against Xanthomonas perforans T4, indicating the long-term stability of supernatants. Example 4:
Testing interspecies and inter-strains antibacterial activity of cell-free supernatants
Following the screening for antimicrobial activity-producing bacteria against Xanthomonas perforans T4, the inhibitory effect of cell-free supernatants was tested within the most active bacterial species and strains (Table 28).
Table 28: Interspecies and inter-strains antibacterial activity of cell-free supernatants
Figure imgf000041_0001
"±" Standard Error of Mean (SEM)
* Diameter of well is 10 mm
Thus, B. amyloliquefaciens subsp. plantarum 71 inhibited the growth of P. polymyxa To99 and P. polymyxa TFr101 showing a clear halo with diameter 26 mm, while these two strains of P. polymyxa showed the antimicrobial activity against B. amyloliquefaciens subsp. plantarum 71 with 25.16 and 29.00 mm growth inhibitory zones, respectively (Table 28).
Inter-strains activity was shown within P. polymyxa. P. polymyxa TFr101 inhibited the growth of P. polymyxa
To99 showing a clear halo with diameter 12.5 mm, whereas P. polymyxa To99 was not capable of inhibiting P. polymyxa TFr101 (Table 28).
Auto-antimicrobial activity was not confirmed for B. amyloliquefaciens subsp. plantarum 71 and P. polymyxa TFr101 , while P. polymyxa To99 showed the biggest auto-antimicronial activity, with a growth inhibition zone 37.16 mm in diameter. Also, P. polymyxa To99 showed a large growth inhibition zone (25.16 mm) against B. amyloliquefaciens subsp. plantarum 71 (Table 28).
Without being bound by theory, these data suggest that cell-free supernatant of P. polymyxa To99 contains compounds (e.g., proteinaceous compounds such as bacteriocins or lantibiotics) produced by the bacteria to inhibit the growth of similar or closely related bacterial strains. Metabolites resembling bacteriocins or lantibiotics in structure would likely kill pathogens by binding to lipids on their cell membranes and making the cells porous. This would cause intracellular fluid to leak out, which would destroy the pathogen. Another example of such a molecule is nisin, produced by bacteria found in dairy products. Normally, lantibiotics are only effective against Gram-positive bacteria, but they can be induced to destroy Gram-negative pathogens such as those that cause plant diseases, by being combined with a chelator like EDTA. It is clear from above experiments that supernatants produced by these active species have wide-spectrum activity against other bacteria and even against bacteria within the same genus and the same species as the active bacteria.
Example 4.1 :
Enhancing the ant\-Xanthomonas activity of P. polymyxa and B. amyloliquefaciens by fermentation in optimized media
4.1.1 Fermentation in optimized media
The precultures of P. polymyxa and B. amyloliquefaciens subsp. plantarum isolates were prepared as follows: 5 μΙ_ of the respective bacterial culture stored at -80°C was inoculated into 3 mL of Tryptic Soy Broth (TSB) and incubated in a rotary shaker at 250 rpm overnight at 30°C. The appropriate aliquots of each culture broth were used to inoculate production media to start with an initial optical density of 0.01 , corresponding to about 8x106 CFU/mL.
With the goal of identifying more optimal nutrient medium and fermentation conditions for enhancing anti- Xanthomonas activity of the metabolites, optimization experiments were performed (data not shown) comparing 10 different nutrient media, as well as different fermentation conditions. For example, the different conditions tested included: various time periods of incubation (48 h, 72 h, 96 h, etc.); different volumes of medium (e.g., 250 mL or 500 mL); and difference Erlenmeyer flask sizes (e.g., 1 L or 2 L flask). The results of these experiments revealed that the optimal media for Bacillus and Paenbacillus isolates were Landy medium and TSB, respectively. Thus, TSB was used as a production medium for P. polymyxa isolates, while Landy's medium was used for cultivation of B. amyloliquefaciens subsp. plantarum isolates. Landy's medium contains: glucose 20 g/L, L-glutamic acid 5.0 g/L, yeast extract 1.0 g/L, K2HP04 1.0 g/L, MgS04 (7H20) 0.5 g/L, KCI 0.5 g/L, CuS04 1.6 mg/L, Fe2(S04)3 1.2 mg/L, MnSO4 0.4 mg/L.
The production of metabolites was carried out in 2 L conical flasks with 500 mL of TSB or 200 mL of Landy's medium, and shaken at 250 rpm at 30°C for 48 h. Culture media were then centrifuged at 25 000 rpm for 1 hour. The pellets were discarded and the supernatants were filtered using stericup vacuum filtration system (0.2 μιτι). The supernatants were collected and stored at -20°C until usage.
4.1.2 knti-Xanthomonas activity of cell-free supernatants following culture in optimized media
Cell-free supernatants of a number of different B. amyloliquefaciens subsp. plantarum isolates (including VFb49, 71 , 237, 16, 33, and 335), as well as 16 different P. polymyxa isolates (including To99, TFM01 , T1 B, 44, 273 and 329) were obtained by culturing in optimized media as described in Example 4.1.1. The supernatants were diluted 10-fold and tested for activity against X. gardneri DC00T7A, X. perforans T4, and X. campestris 901 using a well-diffusion inhibition assay (Obradovic et al., 2002; WO/2012/051699; Lindow et al., 2003). As shown in Figure 10, 10-fold diluted cell-free supernatants of B. amyloliquefaciens subsp. plantarum isolates 71 and VFb49 showed the highest antimicrobial activity amongst the supernatants tested against X. gardneri DC00T7A, X. perforans T4, and X. campestris 901. Ten-fold diluted supernatants of B. amyloliquefaciens subsp. plantarum isolates 237, 16, and 33 were about 2-fold less active against all thee Xanthomonas species tested, while 10-fold diluted supernatant of B. amyloliquefaciens subsp. plantarum 335 showed no detectable anW-Xanthomas activity in this assay.
Among the 16 P. polymyxa isolates that were tested in the Example, 10-fold diluted supernatants of 6 isolates (To99, TFr101 , T1 B, 44, 273 and 329) showed the highest anW-Xanthomonas activity (Figure 11). Based on these results, B. amyloliquefaciens subsp. plantarum isolates VFb49 and 71 , as well as P. polymyxa isolates To99, TFr101 , and 273 were selected for further characterization of their metabolites.
Example 5:
Efficacy of bacterial cell-free supernatants and their live cells in controlling tomato bacterial spot disease caused by Xanthomonas species
5.1 Bacterial cell-free supernatants
The five bacterial isolates from Examples 4 and 4.1 that showed the highest activities against Xanthomonas species were used in this example: Bacillus amyliquefaciens subsp. plantarum isolates 71 and VFb49, and Paenibacillus polymyxa isolates To99, 273, and TFr101. Of note, the pathogen growth inhibition zone caused by cell-free supernatants of these strains was more than 30 mm in diameter (Table 3, Figure 5), or the inhibition area of pathogen caused by 10-fold diluted bacterial supernatant was more than 200 mm2. This result is comparable to the efficacy of copper plus mancozeb (2 g/L and 1 g/L, respectively; diameter of growth inhibition zone: 28.41 ±0.51 ; see Figure 8), which is used by growers to control bacterial spot disease caused by Xanthomonas species. Production of secondary metabolites by Bacillus and Paenibacillus isolates for testing their efficacy in controlling tomato bacterial spot disease was performed as described above in Example 4.1.1.
5.2 Plant pathogens
Bacterial spot of tomato is one of the most pervasive diseases that faces tomato production all over the world. It is caused by four closely related species of Xanthomonas: X. gardneri, X. euvesicatoria, X. vesicatoria, and X. perforans. After a number of early revisions, they were classified for some time as Xanthomonas campestris pv. vesicatoria (Xcv) [Dye, 1978]. Comparative studies of whole genome sequences from reference strains, as well as data of carbon utilization, amylolitic and pectolytic activities of these four species showed considerable diversity between these pathogens, especially the difference between X. gardneri and other three Xanthomonas species [Potins et al., 2011 ; EPPO Bulletin, 2013]. Also, Race T1 , caused by X. euvesicatoria was the endemic race in Florida until 1991 , when X. perforans race T3 (antagonistic to race T1), emerged. Race T4 came about as a result of a mutation in the X. perforans avrXv3 gene, and has recently become prevalent [Jones et al., 2004; Jones et al., 2005]. Thus, X. perforans race T4 and X. gardneri DC00T7A were used herein.
X. perforans T4 or X. gardneri DC00T7A was incubated in TSB at 30°C for 16h (overnight). Cell culture medium was centrifuged at 10,000xg for 5 min. The supernatant was discarded and bacterial cells were resuspended in sterile distilled water. The NanoDrop™ ND-1000 spectrophotometer was set to measure absorbance at 600 nm wavelength, and the suspension was adjusted to an optical density (ODeoonm) of 0.2, which was empirically determined to represent 2x108 CFU/mL by plating serial dilutions of the suspension and counting colonies. Prior to spraying tomato seedlings, cell suspension of pathogen was mixed with the surfactant Silwet L-77 at 0.025% (v/v) to help in the penetration and infection of the plants.
5.3 Plant materials
Tomato Solanum lycopersicum L. var. cerasiforme (Dunal) (D. M. Spooner et al.) cv. Bonny Best (OSC Seeds, Waterloo, Ontario, Canada) or Florida 47 (Harris seeds, Rochester, NY, US) seeds were planted in Pro-mix™ BX Mycorrhizae™ (Premier horticulture Inc., Quakertown, Pennsylvania, USA) with adding all-purpose NPK (20-20- 20) fertilizer (Plant Products Co. LTD., Brampton, Ontario, Canada).
Tomato seedlings were grown in plastic pots (6.0x6.0 cm) in a growth chamber (25°C, relative humidity (RH) 40%, with a photoperiod of 16 hours (light intensity is about 200.0 lum/sqf) for two weeks (the four-true-leaf stage) prior to infestation by X. perforans T4 or . gardneri DC00T7A.
Temperature, relative humidity (RH), and lightness were monitored hourly during the research using Hobo™ digital system (Onset computer corporation) that was located in the center of the shelf with tomatoes.
5.4 Experimental design
The trials under growth chamber conditions were carried out on tomato seedlings to assess the efficacy of cell-free bacterial supernatants of B. amyliquefaciens subsp. plantarum and P. polymyxa isolates in controlling artificial infections caused by X. perforans T4, as compared to a mix of copper and mancozeb (2 and 1 g/L, respectively).
Each treatment was replicated 3 times with four seedlings per replicate, according to a randomized blocks design. The same number of not-inoculated and treated by water, not-inoculated and treated by TSB plants served as negative controls. Inoculated and treated by water, inoculated and treated by TSB tomato seedlings served as positive controls. Bacterial cell-free supernatants from each strain were applied for testing their phytotoxic effect in plants.
Tomato seedlings were sprayed on abaxial and adaxial leaf surfaces to leaf wetness with a hand sprayer, applying approximately 2 ml_ of X. perforans T4 suspension per plant. After 1 h of inoculation, seedlings were treated by spraying cell-free supernatants, water, or TSB, respectively, using a hand-pump up to run-off of plants. Tomato seedlings were maintained for 48h under plastic bags and then placed in a growth chamber (30°C, RH 60% with a photoperiod of 16 hours) for one week.
The plants were assessed for disease severity by visual estimation of the percentage of leaf tissue with spots and lesions 10 days after infestation. Symptoms on the leaves were recorded for each plant by two persons independently. Disease severity assessments were made based on leaf rating compiled from three separate experiments.
5.5 Curative effect of cell-free bacterial supernatants
Reduction of leaf spots
All treatments with bacterial cell-free supernatants were effective in controlling leaf spot caused by X. perforans T4 on tomato seedlings (Figure 9 A-R).
Thus, severity disease was evaluated as 70% in plants infected by X. perforans T4 and treated by water, and 90% in plants infected by X. perforans T4 and treated by TSB, comparative to not-infected tomato seedlings (Figure 9 A-D). Susceptible reactions manifested 7 days after infestation as small, greasy water-soaked spots (about 1/8 inch) on leaflets. Older spots became dry and brown, and often surrounded by yellow halos. Spots increased in size to form large, irregular dead spots. Lesions were frequently surrounded by large chlorotic haloes and perforations, referring to the holes in the leaf following infection by the bacterium (Figure 9 A-L, O-R).
Treatment with bacterial cell-free supernatant of B. amyloliquefaciens subsp. plantarum isolates 71 (Figure 9 G-H) and VFb49 (Figure 9 O-P), as well as P. polymyxa isolates TFr101 (Figure 9 K-L) and 273 (Figure 9 Q-R) reduced disease severity by almost 4 times compared to the water-treated control (Figure 9 C, D). Thus, only 20% of leaf surface was infected by X. perforans T4 after tomato treatment by cell-free supernatants of these isolates (Figure 9 G, H, K, L, O, P, Q, R).
Almost the same effect in controlling bacterial spot disease was shown for cell-free supernatants of B. amyloliquefaciens subsp. plantarum VFb49 (Figure 9 O, P) and P. polymyxa 273 (Figure 9 Q, R), reducing disease severity by about 4.6-fold as compared to the water-treated control. Only 40% of leaf surfaces of tomato seedlings treated by metabolites of these isolates were infected by X. perforans T4.
The cell-free supernatant of P. polymyxa To99 reduced disease severity by about 2-fold as compared to positive control (Figure 9 I, J). Thus, about 40% of leaf surfaces of tomato seedlings were infected by X. perforans T4, which is comparable with treatment by copper and mancozeb (Figure 9 C-F, I, J). Of note, spots and lesions appeared on only one of six leaves per plant infected by X. perforans T4 and treated by bacterial cell-free supernatants, while infected and not-treated plants had four of six leaves with these disease symptoms.
Bacterial cell-free supernatant of all strains sprayed as is with no dilution did not cause any phytotoxic effect in tomato plants (e.g., Figure 9 M, N). In conclusion, cell-free supernatants of bacterial isolates showed suppression of bacterial spot disease caused by X. perforans T4, comparable to copper-mancozeb, suggesting that they may be potentially suitable replacements to chemical biocides. 5.6 Preparation of live cell suspensions of bacterial isolates and application to tomato leaves
P. polymyxa and B. amyloliquefaciens subsp. plantarum isolates were removed from frozen stock vials and 5 μΙ_ of each isolate was added to 3 ml_ of TSB. Bacteria were cultivated at 30°C for 16h (overnight). Cell culture medium was centrifuged at 10,000 x g for 5 min and washed twice by 0.85% NaCI. The supernatant was discarded and bacterial cells were resuspended in sterile 0.85% NaCI adjusting to the final concentration 3x108 CFU/mL (OD=0.3). Live cell suspension of bacterial isolates was applied on tomato leaves immediately after preparing.
Foliar treatment of tomato seedlings was performed by suspension of live bacterial cells (3 x 108 CFU/mL) of Bacillus and Paenibacillus isolates, and respective control solutions using a hand trigger sprayer with application on abaxial and adaxial leaf surfaces until run-off. To confirm the presence of live bacterial isolates on tomato leaves, leaf print on Tryptic Soy Agar (TSA) plates and serial dilution method were performed 1 hour and 6 days after treatment.
For the leaf print method, one tomato leaf from each plant treated by a respective bacterial isolate was cut and put directly onto TSA plate. Plates were incubated overnight in the dark at 30°C. Then leaves were removed and plates were incubated at 30°C for 48h. Figure 12 shows a typical print of an untreated tomato leaf in panel (A), and one treated with B. amyloliquefaciens subsp. plantarum 71 panel (B) after 24 hours of incubation.
For the serial dilution method, one leaf from each plant was cut onto segments (0.5 cm2), placed in tubes containing 5 mL of 0.85% NaCI and 0.01% Triton-100, resuspended by vortexing for 5 min. Then, 100 μί of each dilution (102, 10"3 and 10 ) were spread on TSA plates for isolation of bacteria. Plates were incubated in the dark at 30°C for 2 days. Quantity of grown colonies was counted and CFU/leaf was calculated. Isolation of microorganisms was performed in triplicate. 5.7 Microbial viability of bacterial isolates on tomato leaves
The leaf print method used in this study (Example 5.6) allowed us to confirm the formation of bacterial biofilm on tomato leaves. Figure 13 shows a whitish film on tomato leaves formed by Bacillus and Paenibacillus isolates 6 days after respective treatments (panels A, C, E, G, I), as well as leaf shape of their live bacterial colonies after leaf printing (panels B, D, F, H, J).
Bacterial cells of all Bacillus and Paenibacillus isolates tested in this Example were still alive on tomato leaves even 6 days following treatments, but the quantity of CFUs per leaf was different depending on the isolates. As shown in Figure 14, 1 hour after treatment, the leaves treated by B. amyloliquefaciens subsp. plantarum 71 contained the highest quantity (log of CFU/leaf reached 3.77) of bacterial live cells, while the quantity of P. polymyxa 273 live cells was 1.6-fold less (log of CFU/leaf reached up 2.33). Six days after treatment, the quantity of CFU/leaf of Bacillus and Paenibacillus isolates increased by about 1.5 fold. There was no significant difference in the quantity of live bacterial cells on untreated tomato leaves after 1 hour and 6 days (Figure 14, "untreated"), while the CFU/leaf of X. gardneri DC00T7A was 1.3-fold higher after 6 days of inoculation (Figure 14, "X. gardneri DC00T7A").
Of note, the colonies formed on TSA by bacteria isolated from untreated tomato leaves were morphologically different from those treated by Bacillus and Paenibacillus isolates. Using the serial dilution method (Example 5.6), 2 fungal and 4 bacterial morphologically different colonies were isolated from untreated tomato leaves after 6 days (Figure 15C), while only B. amyloliquefaciens subsp. plantarum 71 and P. polymyxa To99 colonies appeared on TSA after their isolation from respectively treated tomato leaves (Figure 15A, B). 5.8 Preventive effect of bacterial live cells and their supernatants in controlling bacterial spot disease caused by X. gardneri DC00T7A
Tomato seedlings were sprayed on abaxial and adaxial leaf surfaces to leaf wetness with a hand sprayer, applying approximately 2 ml_ of cell-free bacterial supernatants or live cell suspension per plant. Water and TSB were used as controls. After treatment, plants were placed in a growth chamber (30°C, RH 60% with a photoperiod of 16 hours) for one week. Then, tomato seedlings were infected by X. gardneri DC00T7A in the same way using a hand- pump up to run-off of plants and placed in a growth chamber. After 10 days, the effectiveness of cell-free supernatants and live cells was evaluated as the reduction of spot numbers per plant compared to the pathogen-only control. The results were compiled from three separate experiments.
In contrast to yellow-brownish lesions caused by X. perforans T4 on tomato leaves, susceptible reaction caused by X. gardneri DC00T7A manifested 10 days after infestation as well-defined brown spots appeared on leaves (Figure 16 A, B) and stems (Figure 16 C, D). Brown spot are indicated with arrows. Thus, the preventive effect of live cells of B. amyloliquefaciens subsp. plantarum (isolates 71 and VFb49) and P. polymyxa (isolates To99, Tfr101 and 273) and their metabolites was evaluated as the reduction of spot numbers per plant.
As shown in Figure 17, treatment of the leaves of tomato seedlings with live cells of Bacillus and Paenibacillus isolates (as well as by their metabolites) 6 days before X. gardneri DC00T7A infection reduced bacterial spot disease by about 2-5 fold as compared to untreated plants. The metabolites of B. amyloliquefaciens 71 and P. polymyxa 273 were twice more effective in controlling this disease than their live cells, while metabolites of P. polymyxa To99 were 1.6 fold less effective than their live cells. There was no statistical difference between the efficacy of live cells and their metabolites of P. polymyxa TFr101 and B. amyloliquefaciens VFb49 in reduction of bacterial spots caused by X. gardneri DC00T7A. All Bacillus and Paenibacillus isolates (live cells as well as metabolites) showed 2-fold suppression of bacterial spot disease caused by X. gardneri DC00T7A, as compared to coppenmancozeb. Example 6:
Characterization of secondary metabolites produced by Bacillus and Paenibacillus isolates
Cell-free supernatants of the B. amyloliquefaciens subsp. plantarum isolates VFb49 and 71 , as well as the P. polymyxa isolates To99, TFr101 , 273 and 329) were obtained by culturing in optimized media as described in Example 4.1.1. 6.1 Sensitivity of metabolites to light exposure
An agar disc diffusion assay was performed in order to evaluate the sensitivity of bacterial metabolites to light exposure. Paper blank discs (diameter = 6 mm) were saturated with 10-fold diluted cell-free supernatants (20 μΙ_), air-dried overnight in the biological cabinet, and exposed to light at room temperature (22°C) for 3 months. Antimicrobial activity was then tested weekly by placing the saturated discs on a lawn of Xanthomonas gardneri DC00T7A and measuring the inhibition area (in mm2). The results are shown in Figure 18 A and B.
As shown in Figure 18A-B, the anW-Xanthomonas activities of the 10-fold diluted cell-free supernatants of almost all of the tested isolates gradually decreased with increasing exposure to light. The cell-free supernatants exhibiting the highest resistance to light exposure were those from the tested strains of Paenibacillus polymyxa, whose activity began to decrease only after 3 weeks of light exposure. In fact, the P. polymyxa-demed supernatants exhibited activity against X. gardneri DC00T7A even after 12 weeks of light exposure, in contrast to those from the B. amyloliquefaciens subsp. plantarum strains. Among the supernatants from the different Paenibacillus polymyxa strains tested, P. polymyxa To99 exhibited the highest activity against X. gardneri DC00T7A.
6.2 Thermal stability of metabolites
To determine the thermal stability of the metabolites, aliquots of each cell-free supernatants were heated at
40°C, 60°C, 80°C or 100°C for 30 min, or autoclaved (121 °C) for 15 min (Kabore et al., 2012; Meng et al., 2012; Compaore et al, 2013). The aliquots of each supernatant were also exposed to 4°C and room temperature (about 22°C) for 6 weeks, and to -20°C for 1 year. The antimicrobial activity was then tested by the agar-well diffusion method by measuring the inhibition area (mm2) of Xanthomonas gardneri DC00T7A, as described in Example 6.1. The results are shown in Tables 29 and 30. Table 29. Effect of heating on the antimicrobial activity of 10-fold diluted Bacillus and Paenibacillus cell-free
supernatants against X. gardneri DC00T7 A
Figure imgf000049_0002
"±" Standard Error of Mean (SEM)
As shown in Table 29, 10-fold diluted cell-free supernatants from all the tested Paenibacillus and Bacillus isolates were at least somewhat sensitive to heating, as their anti-Xanthomonas activities decreased with increasing temperature. Those most sensitive to high temperatures were the cell-free supernatants from the P. polymyxa isolates and B. amyloliquefaciens subsp. plantarum VFb49, which completely lost their activities after autoclaving at
121 °C for 15 minutes. In contrast, 10-fold diluted cell-free supernatant of B. amyloliquefaciens subsp. plantarum 71 was still active against X. gardneri DC00T7A even after autoclaving.
Table 30. Effect of storage of Bacillus and Paenibacillus cell-free supernatants at different temperatures on their
antimicrobial activity against X. gardneri DC00T7 A
Figure imgf000049_0003
"±" Standard Error of Mean (SEM)
* Inhibition area represent the effect of 10-fold diluted supernatants
Values in bold are significantly different (at the indicated p value) from the corresponding control value. The control is the
Figure imgf000049_0001
activity of fresh supernatants after 1 h storage at 4°C and 22°C, respectively.
The antimicrobial activities of Paenibacillus and Bacillus 10-fold diluted cell-free supernatants against X. gardneri DC00T7A after storage at -20°C are shown in Figure 19. The most resistant to the storage at -20°C were metabolites of P. polymyxa To99 and B. amyloliquefaciens subsp. plantarum VFb49. There was no statistically significant difference (p < 0.05) between anti-Xanthomonas activity of P. polymyxa To99 supernatants, even after 6 and 12 months comparing to control. The control was the
Figure imgf000050_0001
activity of fresh supernatant that was tested against Xanthomonas on the same day when it was obtained; and it was not subjected to any of the indicated conditions (i.e., 4°C, 22°C and -20°C).
6.3 Extraction of secondary metabolites and their effects on anti-Xanthomonas activity
Extraction of secondary metabolites by butanol allowed for the detection of particular types compounds (e.g., lipopeptides) produced by Bacillus and Paenibacillus isolates. This method was performed according to Wulf et al. (2002) and Yokota et al. (2012) with some modifications. Briefly, 2 ml_ of culture supernatant was extracted using 2 ml_ of 2-butanol by vortex mixing for 20 seconds. After centrifugation at 10 000 x g for 5 min at 20°C, the organic (upper) layer was collected into glass vials. The remaining aqueous layer was extracted twice adding 500 μΙ_ of 2- butanol. The organic fractions were combined and evaporated at 30°C under a gentle stream of nitrogen. The dried sample was dissolved in 2 ml_ of dhbO. The antimicrobial activity of undiluted and 10-fold diluted fractions was estimated using well diffusion method by measuring the inhibition area (mm2) of Xanthomonas gardneri DC00T7 A, as described in Example 6.1. The results are shown in Table 31.
Table 31. Antimicrobial activity of Bacillus and Paenibacillus metabolite extractions against X. gardneri DC00T7A
Figure imgf000050_0002
The anti-Xanthomonas activity of organic and aqueous phases of all 5 bacterial isolates indicates that they produce several compounds, including lipopeptides, with different diffusion abilities. For example, 100% organic phase (lipopeptides) of P. polymyxa To99 supernatant showed the same anW-Xanthomonas activity as 100% crude supernatant, while 100% aqueous phase of its supernatant was 1.2-fold more active against X. gardneri DC00T7A. Regarding P. polymyxa isolates TFr101 and 273, the aqueous phases of their supernatants were more active against X. gardneri DC00T7A than their organic phases. In contrast, the aqueous phases of Bacillus supernatants were less active against X. gardneri DC00T7A than their corresponding organic phases. Among all 5 bacterial isolates, the organic phase of B. amyloliquefaciens subsp. plantarum VFb49 supernatant, as well as its crude supernatant, showed the highest anti-Xanthomonas activity (Table 31). Example 7:
Complete genome sequencing
The whole genome sequencing was performed to further confirm the taxonomic identification of the isolates, in addition to the results already obtained from the sequencing of 16S rDNA (Example 2.2) and specific {gyrA, gyrB and rpoB) genes (Examples 2.3 and 2.5), biochemical tests of Biolog™ microbial identification system (Example 2.3a), and fatty acid analyses (Example 2.4).
7.1 Materials and Methods
Genomic DNA was isolated from an overnight culture of each strain (71 , VFb49, To99, TFr101 , and 273) using a Qiagen DNeasy™ blood and tissue kit (Qiagen Inc., Valencia, CA). Genome sequencing was performed using lllumina MiSeq™ sequencing system (lllumina, San Diego, CA), achieving >50x average genome coverage. De novo assembly was created for each genome using SPAdes™ 3.0.0 (St. Petersburg genome assembler), and annotated with the NCBI Prokaryotic Genomes Automatic Annotation Pipeline (http://www.ncbi.nlm.nih .gov/genomes/static/Pipeline.html). Taxonomy of each strain was assigned using Kraken™ (Wood and Salzberg, 2014), a metagenomics sequence classification tool (http://ccb.jhu.edu/software/kraken/). Genome mining of biosynthetic gene clusters, including non-ribosomal peptide synthetases (NRPSs) and other secondary metabolites, were predicted with antibiotics & Secondary Metabolite Analysis SHell™ (antiSMASH™) (Weber et al., 2015) web server (http://antismash.secondarymetabolites.org/). 7.2 Results
Bacillus amyloliquefaciens subsp. plantarum isolates 71 and VFb71 , as well as Paenibacillus polymyxa isolates To99, TFr101 and 273 possessing the highest anti-Xanthomonas activity were subjected to the whole genome sequence analysis. Genome annotations for these bacterial isolates are summarized below.
High genome coverage (88-119%) and very low (2.06-10.47%) percentage of unclassified reads of all isolates reflect the accuracy of the species identification (Table 32.1).
Table 32.1. Genome se uencin and annotation details of the most active isolates a ainst Xanthomonas erforans
Figure imgf000051_0001
The ANI calculator was used (http://enve-omics.ce.gatech.edu/ani/index) to compare the nucleotide identity of the bacterial isolates with already known and type strains of Paenibacillus and Bacillus species from the NCBI database (http://www.ncbi.nlm.nih.gov/nuccore). The ANI calculator estimates the average nucleotide identity using both best hits (one-way ANI) and reciprocal best hits (two-way ANI) between two genomic datasets, as calculated by Goris et al., 2007. Typically, the ANI values between genomes of the same species are above 95%. Table 32.2. The average nucleotide identity (%) based on complete genome sequencing between Bacillus (A) and Paenibacillus (B) isolates possessing anW-Xanthomonas activity and type strains
A
Figure imgf000052_0001
B
Figure imgf000052_0002
Thus, the VFb49 and 71 isolates showed about 99% nucleotide identity with Bacillus amyloliquefaciens subsp. plantarum (Table 32.2A), which confirms our previous identification based on sequencing of gyrA, gyrB and rpoB genes, specific for Bacillus subtilis group.
Comparing complete genome sequencing of Paenibacillus isolates To99, TFR101 and 273 with two closely related and well known strains Paenibacillus peoriae HS311 and Paenibacillus polymyxa SC2, we observed that nucleotide identity of both isolates To99 and 273 with Paenibacillus peoriae strain HS311 reached 99%, while isolate TFr101 showed 95% identity with this strain (Table 32.2B). All three Paenibacillus isolates showed only 89% nucleotide identity (<95%) with Paenibacillus polymyxa SC2. Thus, Paenibacillus isolates To99, 273 and TFr101 are more accurately re-identified as Paenibacillus peoriae, instead of Paenibacillus polymyxa.
Comparing complete genome sequencing of Bacillus and Paenibacillus isolates between themselves showed that isolates To99 and 273 are closely related isolates with nucleotide identity of 99.20%, while isolate TFr101 was different from both of them and its nucleotide identity with these isolates averaged only 95%. Taking into account that the ANI value between isolate TFr101 and Paenibacillus peoriae strain HS311 is on the border of 95%, probably Paenibacillus isolate TFr101 could be a new species, or it can be explained by high value (10.47%) of unclassified reads.
The antibiotics Secondary Metabolite Analysis Shell (antiSMASH™) tool allowed us to identify 3 polyketide synthetases (PKSs), 15 non-ribosomal peptide synthetases (NRPSs), and 3 hybrid PKSs/NRPSs genes that could be involved in the synthesis of secondary metabolites of Bacillus amyloliquefaciens subsp. plantarum and Paenibacillus polymyxa isolates possessing the highest anW-Xanthomonas activity (Table 32.3). Genome of S. amyloliquefaciens subsp. plantarum (isolates 71 and VFb49) and P. polymyxa (isolates To99, TFr101 and 273) harboured different gene clusters responsible for the production of polyketides, lipopeptides, bacteriocins and lantibiotics putatively involved in controlling bacterial leaf spot caused by Xanthomonas species (Table 32.3). Table 32.3: Presence of genes belonging to biosynthetic clusters encoding secondary metabolites of Bacillus
Figure imgf000053_0001
absence or no detection of the gene cluster; "+": presence or detection of the gene cluster
For example, the genome of B. amyloliquefaciens subsp. plantarum 71 resulted in the identification of gene clusters for the synthesis of polyketides such as bacillaene, difficidin, macrolactin. It is interesting to note that B. amyloliquefaciens subsp. plantarum VFb49 does not possess gene cluster encoding macrolactin production, while bacillaene and difficidin gene clusters were revealed in its genome. Among gene clusters involved in polyketide production by P. polymyxa isolates, the genes clusters responsible for the synthesis of bacillaene and nosperin were revealed in P. polymyxa isolates To99 and 273. The genes encoding kalimantacin/batumin and myxovirescin were discovered in B. amyloliquefaciens subsp. plantarum 71 and P. polymyxa 273 genomes (Table 32.3).
Interestingly, only the genomes of B. amyloliquefaciens subsp. plantarum isolates 71 and VFb49 resulted in the identification of gene clusters responsible for the synthesis of non-ribosomal lipopeptides (NRPs) such as surfactin, fengycin, plipastatin (only for VFb49 isolate), iturin, bacilysin, bacillibactin, locillomycin (only for VFb49 isolate), while the genome of P. polymyxa isolates does not possess these gene clusters. In contrast, all three isolates (To99, TFr101 and 273) of P. polymyxa possess gene cluster responsible for the synthesis of NRPs such as polymyxin and fusaricidin. Gene clusters involved in bacillomycin and pelgipeptin biosynthesis were found in P. polymyxa isolates To99 and TFr101. Gene cluster responsible for tridecaptin and bacitracin synthesis was discovered in the genome of P. polymyxa isolates TFr101 and 273. Only the genome of P. polymyxa To99 possesses gene clusters involved in synthesis of paenilarvin and paenibacterin..
Gene clusters responsible for the peptide biosynthesis by RPSs such as plantathiazolicin/plantazolicin and bacteriocins were detected only in the genome of B. amyloliquefaciens subsp. plantarum 71. Among gene clusters involved in the biosynthesis of lantipeptides, B. amyloliquefaciens subsp. plantarum VFb49 possesses gene cluster for subtilin, while gene clusters encoding paenicidin B and paenibacillin biosynthesis are found in the genome of P. po/ymyxa TFr101 (Table 32.3).
The genomes of both isolates (71 and VFb49) of B. amyloliquefaciens subsp. plantarum possessing anti-
Xanthomonas activity harbour gene clusters involved in the biosythensis of cyclic lipopeptides such as surfactin, fengycin, plipastatin and iturin; as well as of the siderophore, bacillibactin. The isolate VFb49 also harbour gene clusters involved in the biosythensis of locillomycin, a new family of cyclic lipopeptides (Luo et al., 2015).
Example 8:
Greenhouse Trials
Two greenhouse trials were conducted to determine the efficacy of metabolites and/or live bacterial strains to control bacterial leaf spots, as compared to standard chemicals agents. The two greenhouse trials were conducted by FarmForest at the University of Ottawa, the first in 2014 and the second in 2015. In general, each plot consisted of at least 6 tomato plants raised from seed in the greenhouse. Each treatment was replicated at least 4 times (24 plants per treatment total). The plants were maintained in conditions similar to those of commercial greenhouse practice, with the appropriate use of fertilizers and insecticides. Plants were inoculated with a virulent strain of Xanthomonas gardneri, two days after the initial application of the treatments. The copper fungicide (Kocide™ 3000) was used at 1.68 kg/ha + Tanos at 0.42 kg/ha with 200 L/ha water volume. Initial treatments were applied 2 DBI as foliar spray, followed by foliar spray applications every 7 days +/- 1 day for 4 more weeks for a total of 5 applications.
The results of the two greenhouse trials are shown below in Tables 33-35 and Figures 20-23. "LIVE" indicates that live strains were administered instead of only metabolites. In general, the results show that all treatments, whether metabolites or live strains, had an effect in controlling the Xanthomonas gardneri and were much better than untreated or Mock (Media used for growth). In some case, the treatments were even equal or better than Copper/Tanos standards. Figure 20 shows the disease control percentage of the metabolites compared to Copper/Tanos mixture in both trials. Figure 21 shows the disease control percentage of the live isolates compared to Copper/Tanos mixture in both trials. Figure 22 shows the AUDPC disease control percentage of the metabolites compared to Copper/Tanos mixture in both trials. Figure 23 shows the AUDPC disease control percentage of the live isolates compared to Copper/Tanos mixture in both trials.
Table 33: Efficacy of the treatments compared to Copper/Tanos mixture in two consecutive greenhouse trials
Figure imgf000055_0001
Table 34: Details of the first trial conducted by FarmForest at the University of Ottawa
Figure imgf000055_0002
S. amyloliquefaciens subsp. plantarum 71 4.8 c 73 56.1 b e d 72
B. amyloliquefaciens subsp. plantarum VFB49
P. peoriae 273
P. peoriae To99 - LIVE 4.6 c 74 42.3 c d 79
B. amyloliquefaciens subsp. plantarum 71 - LIVE 6.7 b e 62 119.6 be 41
B. amyloliquefaciens subsp. plantarum VFb49 - LIVE
P. peoriae 273 - LIVE
In the Table above, the letters a, b, c, and d indicate statistical significance.
Table 35: Details of the second trial conducted by Farm Forest at the University of Ottawa
Figure imgf000056_0001
The present description refers to a number of documents, the content of which is herein incorporated by reference in their entirety. The scope of the claims should not be limited by the preferred embodiments set forth in the examples, but should be given the broadest interpretation consistent with the description as a whole.
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Claims

CLAIMS:
1. A composition comprising metabolites from a bacterial and/or fungal species, wherein said metabolites have antimicrobial activity against Xanthomonas species.
2. The composition of claim 1 , wherein said metabolites are extracellular bacterial and/or extracellular fungal metabolites.
3. The composition of claim 1 or 2, wherein said metabolites are from a: Bacillus species; Paenibacillus species; Burkholderia species; Mortierella species; Giberella species; Fusarium species; Aspergillus species;
Penicillium species; or any combination thereof.
4. The composition of claim 1 or 2, wherein said metabolites are from: Paenibacillus polymyxa; Paenibacillus peoriae; Bacillus amyloliquefaciens; Burkholderia cepacia; Mortierella alpine; Giberella moniliformis; Fusarium oxysporum; Aspergillus n/gerTiegh; Aspergillus hiratsukae; Penicillium ochrochloron; or any combination thereof.
5. The composition of claim 1 or 2, wherein said metabolites are from Burkholderia cepacia, Paenibacillus polymyxa, Paenibacillus peoriae, and/or Bacillus amyloliquefaciens.
6. The composition of claim 1 or 2, wherein said metabolites are from Paenibacillus peoriae.
7. The composition of claim 1 or 2, wherein said metabolites are from Bacillus amyloliquefaciens.
8. The composition of claim 1 or 2, wherein said metabolites are from: Paenibacillus polymyxa T1 B; Paenibacillus polymyxa 44; Paenibacillus sp. 62; Paenibacillus peoriae 273; Paenibacillus polymyxa 329;
Paenibacillus sp. 344; Paenibacillus polymyxa 390; Paenibacillus peoriae To99; Paenibacillus polymyxa TP12; Paenibacillus polymyxa TP29; Paenibacillus polymyxa TP77; Paenibacillus polymyxa V25T; Paenibacillus polymyxa TFr60; Paenibacillus sp. TFr101 ; Paenibacillus polymyxa TAu1; Paenibacillus polymyxa TM54; Bacillus amyloliquefaciens subsp. plantarum 16; Bacillus amyloliquefaciens subsp. plantarum 33; Bacillus amyloliquefaciens subsp. plantarum 71; Bacillus amyloliquefaciens subsp. plantarum 237; Bacillus amyloliquefaciens subsp. plantarum 335; Bacillus amyloliquefaciens subsp. plantarum VFb49; Burkholderia cepacia BC19; Burkholderia cepacia BC153; Mortierella sp. VFb1 ; Giberella sp. TFr4; Fusarium sp. FI3S; Aspergillus sp. 8PT; Aspergillus sp. FG; Penicillium sp. VFr37; or any combination thereof.
9. The composition of claim 1 or 2, wherein said metabolites are from: Bacillus amyloliquefaciens subsp. plantarum 71 (NRRL B-67021); Bacillus amyloliquefaciens subsp. plantarum VFb49; Paenibacillus peoriae To99 (NRRL B-67020); Paenibacillus peoriae TFr101 (NRRL B-67019); Paenibacillus peoriae 273, or any combination thereof.
10. The composition of any one of claims 1 to 9, wherein said metabolites have antimicrobial activity against phytopathogenic Xanthomonas species.
11. The composition of any one of claims 1 to 10, wherein said metabolites have antimicrobial activity against Xanthomonas campestns, Xanthomonas perforans, Xanthomonas gardnen, or any combination thereof.
12. The composition of claim 11 , wherein said Xanthomonas campestns comprises Xanthomonas campestns MAPAQ #901 and/or Xanthomonas campestns ED1985, or wherein said Xanthomonas gardnen comprises Xanthomonas gardnen DC00T7A.
13. The composition of claim 11 , wherein said Xanthomonas perforans comprises Xanthomonas perforans T1 , T2, T3, T4, T5, or any combination thereof.
14. The composition of claim 13, wherein said Xanthomonas perforans comprises Xanthomonas perforans T4.
15. The composition of any one of claims 1 to 14, wherein said metabolites further have antimicrobial activity against a plant and/or a human pathogenic microorganism.
16. The composition of claim 15, wherein said pathogenic microorganism is a virus, bacteria, fungus, yeast, mold, or any combination thereof.
17. The composition of claim 15, wherein said pathogenic microorganism is: Xanthomonas euvesicatoria; Xanthomonas fraganae; Xanthomonas perforans; Xanthomonas campestns; Xanthomonas gardnen; Pseudomonas syringae; Erwinia amylovora; Burkholderia glumae; Escherichia coli; Bacillus subtilis; Staphylococcus aureus; Pseudomonas aeruginosa; or any combination thereof.
18. The composition of claim 17, wherein said pathogenic microorganism is: Xanthomonas euvesicatoria R4; Xanthomonas gardnen DC00T7A, Xanthomonas fragariae LMG 708; Pseudomonas syringae DC3000; Erwinia amylovora 435; Burkholderia glumae LMG10905; Escherichia coli 0157:H7 EDL933; Bacillus subtilis ED66; Staphylococcus aureus ED711; Pseudomonas aeruginosa PA416A; or any combination thereof.
19. The composition of any one of claims 1 to 18, wherein said antimicrobial activity comprises anti-bacterial and/or anti-fungal antagonistic activity.
20. The composition of any one of claims 1 to 19, wherein said metabolites exhibit higher antimicrobial activity against Xanthomonas species, as compared to other species.
21. The composition of any one of claims 1 to 20, wherein said metabolites comprise lipopeptides and/or siderophores having anW-Xanthomonas activity.
22. The composition of claim 21, wherein said lipopeptides and/or siderophores are from Bacillus and/or Paenibacillus.
23. The composition of claim 21 or 22, wherein said lipopeptides are non-ribosomal lipopeptides (NRPs).
24. The composition of claim 21 or 22, wherein said lipopeptides and/or siderophores comprise surfactin, fengycin, plipastatin, iturin, bacilysin, bacillibactin, bacillomycin, locillomycin, paenilarvin, pelgipeptin, polymyxin, paenibacterin, fusaricidin, bacitracin, tridecaptin, or any combination thereof.
25. The composition of claim 21 or 22, wherein said metabolites comprise plipastatin and/or locillomycin.
26. The composition of any one of claims 1 to 25, further comprising an agriculturally acceptable excipient.
27. The composition of any one of claims 1 to 26, further comprising one or more of: non-toxic carriers, surfactants, preservatives, nutrients, UV protectants, stickers, spreaders and chelating agents.
28. The composition of any one of claims 1 to 27, wherein said composition lacks viable cells from said bacterial and/or fungal species.
29. The composition of claim 28, wherein said composition is a cell-free composition.
30. The composition of claim 29, wherein said composition is a cell-free supernatant.
31. The composition of any one of claims 1 to 28, wherein said composition comprises killed cells from said bacterial and/or fungal species.
32. The composition of any one of claims 1 to 28, wherein said composition lacks viable cells from said bacterial and/or fungal species.
33. The composition of any one of claims 1 to 28, wherein said composition comprises viable cells from said bacterial and/or fungal species.
34. The composition of any one of claims 1 to 33, wherein said composition comprises spores from said bacterial and/or fungal species.
35. The composition of any one of claims 1 to 34, wherein said composition is in the form of a liquid, concentrate, powder, tablet, gel, pellets, granules, or any combination thereof.
36. The composition of any one of claims 1 to 35, wherein said composition, once applied to a target plant, has no detectable phytotoxic effect on said target plant, or on the fruits, nuts, or leaves thereof
37. The composition of any one of claims 1 to 36, wherein said composition comprises at least 100 ppm, 200 ppm, 300 ppm, 400 ppm, 500 ppm, 600 ppm, 700 ppm, 800 ppm, 900 ppm, 1000 ppm, 1500 ppm, 2000 ppm , 2500 ppm, 3000 ppm, 3500 ppm, 4000 ppm, 4500 ppm, 5000 ppm, 5500 ppm, 6000 ppm, 6500 ppm, 7000 ppm, 8000 ppm, 8500 ppm, 9000 ppm, or 9500 ppm of said metabolites.
38. The composition of any one of claims 1 to 36, wherein said composition comprises between about 100 ppm, 200 ppm, 300 ppm, 400 ppm, 500 ppm, 600 ppm, 700 ppm, 800 ppm, 900 ppm, 1000 ppm, 1500 ppm, 2000 ppm,
2500 ppm, 3000 ppm, 3500 ppm, 4000 ppm, 4500 ppm, 5000 ppm, to about 10 000 ppm of said metabolites.
39. The composition of any one of claims 1 to 38, which is a biopesticide.
40. The composition as defined in any one of claims 1 to 39, for use as an anti-microbial agent against a plant and/or human pathogenic microorganism.
41. Use of the composition as defined in any one of claims 1 to 39, as an anti-microbial agent against a plant and/or human pathogenic microorganism.
42. Use of the composition as defined in any one of claims 1 to 39, for the manufacture of an anti-microbial agent against a plant and/or human pathogenic microorganism.
43. The composition of claim 40 or the use of claim 41 or 42, wherein said pathogenic microorganism is a Xanthomonas species.
44. The composition of claim 40 or the use of any one of claims 41 to 43, for application to a growing plant.
45. The composition or use of claim 44, wherein said growing plant is a fruit plant, nut, cereal, vegetable, or flower.
46. The composition or use of claim 45, wherein said fruit is: apple, apricot, banana, blackberry, blueberry, cantaloupe, cherry, cranberry, currant, grapes, greengage, gooseberry, honeydew, lemon, mandarin, melon, orange, peach, pears, pineapple, plum, raspberry, strawberry, tomatoes, watermelon, grapefruit, pepper, olive, or lime.
47. The composition or use of claim 45, wherein said nut is: almond, beech nut, Brazil nut, butternut, cashew, chestnut, chinquapin, filbert, hickory nut, macadamia nut, pecan, walnut, or pistachio.
48. The composition or use of claim 45, wherein said cereal is: amaranth, breadnut, barley, buckwheat, canola, corn, fonio, kamut, millet, oats, quinoa, cattail, chia, flax, kahiwa, pitseed goosefoot, wattleseed, rice, rye, sorghum, spelt, teff, triticale, wheat, or colza.
49. The composition or use of claim 45, wherein said vegetable is: artichoke, bean, beetroot, broad bean, broccoli, cabbage, carrot, cauliflower, celery, chicory, chives, cress, cucumber, kale, dill, eggplant, kohlrabi, lettuce, onion, pepper, parsnip, parsley, pea, potato, pumpkin, radish, shallot, soybean, spinach, turnip, or peanut.
50. The composition or use of claim 45, wherein the growing plant is tomato plant.
51 The composition or use of claim 45, wherein the growing plant is pepper plant.
52. The composition or use of claim 45, wherein the growing plant is a berry plant.
53. The composition or use of claim 52, wherein said berry plant is a strawberry plant.
54. The composition or use of claim 45, wherein the growing plant is lettuce.
55. The composition or use of claim 45, wherein the growing plant is a citrus plant.
56. The composition or use of claim 45, wherein the growing plant is a walnut plant.
57. The composition or use of claim 45, wherein the growing plant is a rice plant.
58. The composition or use of claim 45, wherein the growing plant is a kiwi plant.
59. The composition or use of claim 45, wherein the flower is: a species of the Euphorbiaceae; Euphorbia pulcherrima (poinsettia), Euphorbia milii (crown-of-thorns), Codiaeum variegatum (croton); a member of the family
Rosaceae (Rosoideae/Rosa), Begoniaceae (Begonia), Araceae; Dieffenbachia, Anthurium, Philodendreae (Philodendron), Caladieae (Syngonium), English ivy or another Araliaceae species; Pelargonium (geranium), Ficus, Hydrangea, Zinnia, ornamental Prunus species, ornamental Peppers, or another flower and/or ornamental plant susceptible to infection by Xanthamonas.
60. The composition or use of any one of claims 44 to 59, for application to a plant cell or tissue which is: a leaf, a stem, a flower, a fruit, a tuber, a rhizome, a corm, a root, or any combination thereof.
61. A method for producing a composition as defined in any one of claims 1 to 39, said method comprising culturing viable cells from said bacterial and/or fungal species to produce said metabolites; and harvesting said metabolites produced therefrom.
62. The method of claim 61 , wherein said viable cells are cultured in Landy medium or Tryptic Soy Broth (TSB).
63. An antimicrobial composition produced by the method of claim 61 or 62.
64. A method for controlling the growth of a pathogenic microorganism on a target plant or tissue; said method comprising contacting said target plant or tissue with the composition as defined in any one of claims 1 to 39.
65. The method of claim 64, wherein said contacting comprising spraying, irrigating, painting, daubing, and/or fogging, onto and/or into the target plant or tissue, the target plant or tissue's hydroponic substrate, and/or the target plant or tissue's agricultural earth.
66. A kit for preparing an aqueous solution for use in controlling pathogens on a plant tissue of a growing plant, said kit comprising: (a) the organism as defined in any one of claims 3 to 9; and/or the composition as defined in any one of claims 1 to 39; and (b) a suitable container.
67. The kit of claim 66, wherein said container is a pouch, a tablet, or a bucket.
68. The kit of claim 66 or 67 for use in controlling infection by the pathogenic microorganism on a plant.
69. The kit of claim 68, wherein said pathogenic microorganism is a Xanthomonas species.
PCT/CA2016/050571 2015-05-22 2016-05-20 Bacterial and fungal metabolites possessing anti-microbial activity against xanthomonas species, compositions, methods, kits and uses relating to same WO2016187703A1 (en)

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